Subtilase variants

ABSTRACT

The present invention relates to novel protease variants exhibiting improved stability and or improved wash performance in liquid detergent. The variants of the invention are suitable for use in e.g. cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions. The present invention also relates to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.15/534,522, filed Jun. 9, 2017, which is a 35 U.S.C. 371 nationalapplication of PCT/EP2015/079574 filed Dec. 14, 2015, which claimspriority or the benefit under 35 U.S.C. 119 of European application nos.14197966.6 and 14197968.2, both filed Dec. 15, 2014, respectively, thecontents of which are fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to novel subtilase variants exhibitingincreased stability and/or improved wash performance in liquid detergentcompositions. The variants of the invention are suitable for use in e.g.cleaning or detergent compositions, such as laundry detergentcompositions and dish wash compositions, including automatic dish washcompositions. The present invention also relates to isolated DNAsequences encoding the variants, expression vectors, host cells, andmethods for producing and using the variants of the invention.

Description of the Related Art

In the detergent industry, enzymes have for many decades beenimplemented in washing formulations. Enzymes used in such formulationscomprise proteases, lipases, amylases, cellulases, mannosidases as wellas other enzymes or mixtures thereof. Commercially the most importantenzymes are proteases.

A wild type subtilase that have been used in laundry is the BLAPprotease disclosed in WO 91/02792.

An increasing number of commercially used proteases are proteinengineered variants of naturally occurring wild type proteasesEverlase®, Relase®, Ovozyme®, Polarzyme®, Liquanase®, Liquanase Ultra®and Kannase® (Novozymes a/s), Purafast®, Purafect OXP®, FN3®, FN4® andExcellase® (Genencor International, Inc.). Further, a number of variantsare described in the art, such as in WO 2004/041979 (NOVOZYMES A/S)which describes subtilase variants exhibiting alterations relative tothe parent subtilase in e.g. wash performance, thermal stability,stability during wash or catalytic activity. The variants are suitablefor use in e.g. cleaning or detergent compositions.

Variants of the BLAP protease and suitable for use in cleaning ordetergent compositions have been disclosed in e.g. EP 701 605.

WO 99/57155 discloses detergent enzymes such as proteases modified byattachment of a cellulose binding domain to the enzymes. It is suggestedthat binding the detergent enzymes such as protease to textilecontaining cellulose would enhance wash performance.

However, various factors make further improvement of the proteasesadvantageous. In particular liquid detergent compositions remain achallenge for many detergent proteases and loss of activity duringstorage remains a problem for many good detergent proteases. Thusdespite the intensive research in protease development there remains aneed for new and improved proteases that have a satisfactory washperformance and increased stability.

SUMMARY OF THE INVENTION

The invention relates to variant subtilases having improved stabilityand/or improved wash performance in liquid detergents compared with theparent subtilase. The invention relates to a subtilase variant having atleast 90% sequence identity to SEQ ID NO: 3, preferably at least 95%sequence identity, preferably at least 96% sequence identity, preferablyat least 97% sequence identity, preferably at least 98% sequenceidentity to SEQ ID NO: 3, wherein the variant has a glutamic acidresidue (E) in position 101, and where the variant has increasedstability in a liquid detergent composition compared to the subtilasehaving the amino acid sequence of SEQ ID NO: 3. The subtilase furthercomprises a substitution selected from the group consisting of S156D,L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61D,R, S87E,G97S, A98D,E,R, S106A,W, N117E, H120V,D,K,N, S124M, P129D, E136Q, S143W,S161T, S163A,G, Y171L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T,N261T,D and L262N,Q,D.

Definitions

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Detergent composition: The term “detergent composition”, includes unlessotherwise indicated, granular or powder-form all-purpose or heavy-dutywashing agents, especially cleaning detergents; liquid, gel orpaste-form all-purpose washing agents, especially the so-calledheavy-duty liquid (HDL) types; liquid fine-fabric detergents; handdishwashing agents or light duty dishwashing agents, especially those ofthe high-foaming type; machine dishwashing agents, including the varioustablet, granular, liquid and rinse-aid types for household andinstitutional use; liquid cleaning and disinfecting agents, includingantibacterial hand-wash types, cleaning bars, soap bars, mouthwashes,denture cleaners, car or carpet shampoos, bathroom cleaners; hairshampoos and hair-rinses; shower gels, foam baths; metal cleaners; aswell as cleaning auxiliaries such as bleach additives and “stain-stick”or pre-treat types. The terms “detergent composition” and “detergentformulation” are used in reference to mixtures which are intended foruse in a wash medium for the cleaning of soiled objects. In someembodiments, the term is used in reference to laundering fabrics and/orgarments (e.g., “laundry detergents”). In alternative embodiments, theterm refers to other detergents, such as those used to clean dishes,cutlery, etc. (e.g., “dishwashing detergents”). It is not intended thatthe present invention be limited to any particular detergent formulationor composition. The term “detergent composition” is not intended to belimited to compositions that contain surfactants. It is intended that inaddition to the subtilase variants according to the invention, the termencompasses detergents that may contain, e.g., surfactants, builders,chelators or chelating agents, bleach system or bleach components,polymers, fabric conditioners, foam boosters, suds suppressors, dyes,perfume, tannish inhibitors, optical brighteners, bactericides,fungicides, soil suspending agents, anticorrosion agents, enzymeinhibitors or stabilizers, enzyme activators, transferase(s), hydrolyticenzymes, oxido reductases, bluing agents and fluorescent dyes,antioxidants, and solubilizers.

Dish wash: The term “dish wash” refers to all forms of washing dishes,e.g. by hand or automatic dish wash. Washing dishes includes, but is notlimited to, the cleaning of all forms of crockery such as plates, cups,glasses, bowls, all forms of cutlery such as spoons, knives, forks andserving utensils as well as ceramics, plastics such as melamine, metals,china, glass and acrylics.

Dish washing composition: The term “dish washing composition” refers toall forms of compositions for cleaning hard surfaces. The presentinvention is not restricted to any particular type of dish washcomposition or any particular detergent.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Hard surface cleaning: The term “Hard surface cleaning” is definedherein as cleaning of hard surfaces wherein hard surfaces may includefloors, tables, walls, roofs etc. as well as surfaces of hard objectssuch as cars (car wash) and dishes (dish wash). Dish washing includesbut are not limited to cleaning of plates, cups, glasses, bowls, andcutlery such as spoons, knives, forks, serving utensils, ceramics,plastics such as melamine, metals, china, glass and acrylics.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication. Improved wash performance: Theterm “improved wash performance” is defined herein as a subtilasevariant displaying an alteration of the wash performance relative to theparent subtilase (i.e. relative to a subtilase having the identicalamino acid sequence of said variant but excluding the alterations insaid variant), such as relative to the mature polypeptide of SEQ ID NO:2 e.g. by increased stain removal. The term “wash performance” includeswash performance in dish wash but also in laundry. The wash performancemay be determined by calculating the so-called intensity value (Int) asdefined in the Automatic Mechanical Stress Assay (AMSA) for AutomaticDish Wash in the Materials and Methods section herein.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Laundering: The term “laundering” relates to both household launderingand industrial laundering and means the process of treating textilesand/or fabrics with a solution containing a detergent composition of thepresent invention. The laundering process can for example be carried outusing e.g. a household or an industrial washing machine or can becarried out by hand.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent: The term “parent” means a protease to which an alteration ismade to produce the enzyme variants of the present invention. Thus theparent is a protease having the identical amino acid sequence of saidvariant but not having the alterations at one or more e.g. two or moreof said specified positions. It will be understood that in the presentcontext the expression “having identical amino acid sequence” relates to100% sequence identity. The parent may be a naturally occurring(wild-type) polypeptide. In a particular embodiment the parent is aprotease with at least 60% identity, such as at least 65%, at least 70%,at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identity to a polypeptide with themature polypeptide of SEQ ID NO: 2.

Protease: The term “protease” is defined herein as an enzyme thathydrolyses peptide bonds. It includes any enzyme belonging to the EC 3.4enzyme group (including each of the thirteen subclasses thereof). The ECnumber refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press,San Diego, Calif., including supplements 1-5 published in Eur. J.Biochem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J.Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J.Biochem. 1999, 264, 610-650; respectively.

Protease activity: The term “protease activity” means a proteolyticactivity (EC 3.4). Proteases of the invention are endopeptidases (EC3.4.21). There are several protease activity types: The three mainactivity types are: trypsin-like where there is cleavage of amidesubstrates following Arg or Lys at P1, chymotrypsin-like where cleavageoccurs following one of the hydrophobic amino acids at P1, andelastase-like with cleavage following an Ala at P1. For purposes of thepresent invention, protease activity is determined according to theprocedure described in “Materials and Methods” below. The subtilasevariants of the present invention have at least 20%, e.g., at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, and at least 100% of the protease activity of the maturepolypeptide of SEQ ID NO: 2.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”. For purposes of the present invention, the sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 orlater. The parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the—nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Stability: The term “stability” includes storage stability and stabilityduring use, e.g. during a wash process (in wash stability) and reflectsthe stability of the subtilase variant according to the invention as afunction of time e.g. how much activity is retained when the protease iskept in solution, in particular in a detergent solution. The stabilityis influenced by many factors e.g. pH, temperature, detergentcomposition e.g. amount of builder, surfactants etc. The proteasestability may be measured using the ‘stability assay’ as described inthe Materials and Methods section herein. The term “improved stability”or “increased stability” is defined herein as a variant proteasedisplaying an increased stability in solutions, relative to thestability of the parent protease. The terms “improved stability” and“increased stability” includes “improved chemical stability”, “detergentstability” or “improved detergent stability.

The term “improved chemical stability” is defined herein as a variantenzyme displaying retention of enzymatic activity after a period ofincubation in the presence of a chemical or chemicals, either naturallyoccurring or synthetic, which reduces the enzymatic activity of theparent enzyme. Improved chemical stability may also result in variantsbeing more able to catalyze a reaction in the presence of suchchemicals. In a particular aspect of the invention the improved chemicalstability is an improved stability in a detergent, in particular in aliquid detergent. The term “detergent stability” or “improved detergentstability is in particular an improved stability of the proteaseactivity when a protease variant of the present invention is mixed intoa liquid detergent formulation, especially into a liquid detergentformulation according to table 1 and then stored at temperatures between15 and 50° C., e. g. 20° C., 30° C. or 40° C.

The term “improved thermal activity” means a variant displaying analtered temperature-dependent activity profile at a specific temperaturerelative to the temperature-dependent activity profile of the parent orrelative to a protease with SEQ ID NO: 3. The thermal activity valueprovides a measure of the variant's efficiency in enhancing catalysis ofa hydrolysis reaction over a range of temperatures. A more thermo activevariant will lead to an increase in enhancing the rate of hydrolysis ofa substrate by an enzyme composition thereby decreasing the timerequired and/or decreasing the enzyme concentration required foractivity. Alternatively, a variant with a reduced thermal activity willenhance an enzymatic reaction at a temperature lower than thetemperature optimum of the parent defined by the temperature-dependentactivity profile of the parent.

Stringency conditions: The different stringency conditions are definedas follows.

The term “very low stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 35% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C.

The term “low stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 1×SSC, 0.2% SDS at 60° C.

The term “medium stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 1×SSC, 0.2% SDS at 65° C.

The term “medium-high stringency conditions” means for probes of atleast 100 nucleotides in length, prehybridization and hybridization at42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 35% formamide, following standard Southernblotting procedures for 12 to 24 hours. The carrier material is finallywashed three times each for 15 minutes using 0.5×SSC, 0.2% SDS at 65° C.

The term “high stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 0.3×SSC, 0.2% SDS at 65° C.

The term “very high stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 35% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 0.15×SSC, 0.2% SDS at 65° C.

Substantially pure variant: The term “substantially pure variant” meansa preparation that contains at most 10%, at most 8%, at most 6%, at most5%, at most 4%, at most 3%, at most 2%, at most 1%, and at most 0.5% byweight of other polypeptide material with which it is natively orrecombinantly associated. Preferably, the variant is at least 92% pure,e.g., at least 94% pure, at least 95% pure, at least 96% pure, at least97% pure, at least 98% pure, at least 99%, at least 99.5% pure, and 100%pure by weight of the total polypeptide material present in thepreparation. The variants of the present invention are preferably in asubstantially pure form. This can be accomplished, for example, bypreparing the variant by well-known recombinant methods or by classicalpurification methods.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” means a polynucleotide preparation free of otherextraneous or unwanted nucleotides and in a form suitable for use withingenetically engineered polypeptide production systems. Thus, asubstantially pure polynucleotide contains at most 10%, at most 8%, atmost 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, andat most 0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′- and3′-untranslated regions, such as promoters and terminators. It ispreferred that the substantially pure polynucleotide is at least 90%pure, e.g., at least 92% pure, at least 94% pure, at least 95% pure, atleast 96% pure, at least 97% pure, at least 98% pure, at least 99% pure,and at least 99.5% pure by weight. The polynucleotides of the presentinvention are preferably in a substantially pure form.

Variant: The term “variant” means a polypeptide having protease activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more (e.g., several) positions. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; a deletion means removal of the amino acid occupying aposition; and an insertion means adding one or more (e.g. several) aminoacids, e.g. 1, 2, 3, 4 or 5 amino acids adjacent to and immediatelyfollowing the amino acid occupying a position.

Wash performance: The term “wash performance” is used as an enzyme'sability to remove stains present on the object to be cleaned during e.g.wash, such as laundry or hard surface cleaning. The improvement in thewash performance may be quantified by calculating the so-calledintensity value (Int) defined in AMSA assay, as described in Materialsand Methods section.

Wild-Type subtilase: The term “wild-type subtilase” means a proteaseexpressed by a naturally occurring organism, such as a bacterium,archaea, yeast, fungus, plant or animal found in nature. An example of awild-type subtilase is BLAP i.e. the subtilase having the amino acidsequence of SEQ ID NO: 2.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide BPN′disclosed in SEQ ID NO: 1 is used to determine the corresponding aminoacid residue in another protease. The amino acid sequence of anotherprotease is aligned with the mature polypeptide disclosed in SEQ ID NO:1, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the mature polypeptidedisclosed in SEQ ID NO: 1 is determined using the Needleman-Wunschalgorithm as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version ofBLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anotherprotease can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; MAFFT (version 6.857 or later), and EMBOSS EMMA employingClustalW (1.83 or later), using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 1 such that traditional sequence-based comparison fails to detecttheir relationship Q, other pairwise sequence comparison algorithms canbe used. Greater sensitivity in sequence-based searching can be attainedusing search programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs ( ) Evengreater sensitivity can be achieved if the family or superfamily for thepolypeptide has one or more representatives in the protein structuredatabases. Programs such as GenTHREADER ( )utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix or combinatorial extension, andimplementation of these algorithms can additionally be utilized to querystructure databases with a structure of interest in order to discoverpossible structural homologs.

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. Multiple mutations areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Glyl95*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Glyl95*+Ser411*”or “G195*+S411*”.

Insertions. The insertion of an additional amino acid residue such ase.g. a lysine after G195 may be indicated by: Glyl95GlyLys or G195GK.Alternatively insertion of an additional amino acid residue such aslysine after G195 may be indicated by: *195aL. When more than one aminoacid residue is inserted, such as e.g. a Lys and Ala after G195 this maybe indicated as: Glyl95GlyLysAla or G195GKA. In such cases, the insertedamino acid residue(s) may also be numbered by the addition of lower caseletters to the position number of the amino acid residue preceding theinserted amino acid residue(s), in this example: *195aK *195bA. In theabove example, the sequences 194 to 196 would thus be:

Savinase 194 195 196 A - G - L Variant 194 195 195a 195b 196 A -  G  - K   - A - L

In cases where a substitution and an insertion occur at the sameposition, this may be indicated as S99SD+S99A or in short S99AD. Thesame modification may also be indicated as S99A+*99aD.

In cases where an amino acid residue identical to the existing aminoacid residue is inserted, it is clear that degeneracy in thenomenclature arises. If for example a glycine is inserted after theglycine in the above example this would be indicated by G195GG or*195aGbG. The same actual change could just as well be indicated asA194AG or *194aG for the change from:

Savinase 194  195  196 To: A - G - L Variant 194 195 195a 196 A -  G  - G - L 194 194a 195 196

Such instances will be apparent to the skilled person and the indicationG195GG and corresponding indications for this type of insertions arethus meant to comprise such equivalent degenerate indications.

Multiple alterations: Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Glyl95Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.Alternatively multiple alterations may be separated be space or a commae.g. A170Y G195E or A170Y, G195E respectively.

Different alterations: Where different alterations can be introduced ata position, the different alterations are separated by a comma, e.g.,“Arg170Tyr,Glu” represents a substitution of arginine at position 170with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg170Ala”.

Alternatively different alterations or optional substitutions may beindicated in brackets e.g. Arg170 [Tyr, Glu] or Arg170{Tyr, Glu} or inshort R170 [Y,E] or R170 {Y, E}.

Numbering of Amino Acid Positions/Residues

If nothing else is mentioned the amino acid numbering used hereincorrespond to that of the subtilase BPN′ (BASBPN) sequence. For furtherdescription of the BPN′ sequence, see SEQ ID NO: 1 or Siezen et al.,Protein Eng. 4 (1991) 719-737.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to subtilase variants having improved stabilityand/or improved wash performance in liquid detergents. Preferably thesubtilase variant also has good wash performance, more preferred thevariant has improved wash performance compared with the parentsubtilase, SEQ ID NO: 2 or SEQ ID NO: 3.

The parent subtilase may in principle be any natural occurring subtilaseor it may be a modified subtilase generated by methods known in the artsuch as preparation of hybrids or two or more individual subtilases orby substituting, deleting or inserting one or more amino acid residuesin a given subtilase.

Many subtilases have a proven record of good wash performance and thereis an abundance of publications describing such subtilases and their usein laundry or cleaning processes, however, for use in detergent it isalso important that the subtilases have a satisfactory stability indetergent compositions, such as in liquid detergent. It is preferred touse a parent subtilase having a good wash performance in order toprovide variants of such subtilases having improved stability in liquiddetergent.

A preferred parent subtilase according to the invention is the BLAPprotease having the amino acid sequence of SEQ ID NO: 2, or a subtilasehaving at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. Thepreferred parent subtilase may be a subtilase having the amino acidsequence of SEQ ID NO: 2, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 amino acids have been modified comparedwith SEQ ID NO: 2, and wherein each modification is independently asubstitution of one amino acid residue with another amino acid residue,a deletion of an amino acid residue or an insertion of one amino acidresidue.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 2. In another aspect,the parent comprises or consists of amino acids 1 to 269 of SEQ ID NO:2. In another embodiment, the parent is an allelic variant of the maturepolypeptide of SEQ ID NO: 2.

One preferred parent subtilase is the subtilase having the amino acidsequence of SEQ ID NO: 2 with a substitution of the arginine residue ina position corresponding to position 101 of SEQ ID NO 1 to a glutamicacid residue (R101E). The sequence of this preferred subtilase is shownin SEQ ID NO: 3.

Other preferred parent subtilase according to the invention is theprotease having the amino acid sequence of SEQ ID NO: 3, or a subtilasehaving at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3. Inanother aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 3. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 3. In another aspect,the parent comprises or consists of amino acids 1 to 269 of SEQ ID NO:3. In another embodiment, the parent is an allelic variant of the maturepolypeptide of SEQ ID NO: 3.

The preferred parent subtilase may be a subtilase having the amino acidsequence of SEQ ID NO: 3, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 amino acids have been modified comparedwith SEQ ID NO: 3, and wherein each modification is independently asubstitution of one amino acid residue with another amino acid residue,a deletion of an amino acid residue or an insertion of one amino acidresidue.

Further subtilase variants of the invention includes variants having atleast 90% sequence identity to SEQ ID NO: 3, which variant comprises oneor more of the substitutions S156D; L262E; Q137H; 53T; R45E,D,Q; P55N;T58W,Y,L; Q59D,M,N,T; G61D,R; S87E; G97S; A98D,E,R; S106A,W; N117E;H120V,D,K,N; S124M; P129D; E136Q; S143W; S161T; S163A,G; Y171L; A172S;N185Q; V199M; Y209W; M222Q; N238H; V244T; N261T,D; L262N,Q,D, whereinthe positions are numbered according to SEQ ID NO 1.

In a preferred embodiment, the subtilase variants of the invention haveboth improved stability in liquid detergent and improved washperformance in comparison with the subtilase having the sequence of SEQID NO: 3. Examples of such preferred subtilases of this embodimentincludes subtilase variants having at least 90% sequence identity to SEQID NO: 3 and comprising one or more of the substitutions R45E,D,Q; T58L;G61D; S87E; G97S; A98E; S106A; N117E; H120D,K,V; P129D; E136Q, Q137H;S156D; S161T; S163A,G; V199M; M222Q; N261T; L262E,Q N.

The subtilase variants of the invention may have other substitutionse.g. substitutions known in the art to impart a particular beneficialproperty to the subtilase variants. There is an abundance ofsubstitutions in subtilases known in the art and it is contemplated thatsuch known substitutions may be used in the present invention in orderto impart such known beneficial effects to the subtilase variants of theinvention. The subtilase variants of the invention may comprise one ormore further substitutions which may be used in the present invention inorder to impart additional beneficial effects and/or to improve anexisting effect such as stability and wash performance.

Preferred additional mutations includes one or more of the followingsubstitutions V4I , N76D, V104T, N128Q, S141H, R145H, A194P, G195E,V205I , N218Q, A228V, N238E, or S265H.

Particular preferred examples of subtilase variants of the inventionpreferably having improved stability and/or improve wash performance inliquid detergent compared with the subtilase having the amino acidsequence of SEQ ID NO: 3 include variants comprising the amino acidsequences:

SEQ ID NO: 3+S3T,

SEQ ID NO: 3+R45E,D

SEQ ID NO: 3+P55N,

SEQ ID NO: 3+T58W,Y,L,

SEQ ID NO: 3+Q59D,M,N,T,

SEQ ID NO: 3+G61D,R,

SEQ ID NO: 3+S87E,

SEQ ID NO: 3+G97S,

SEQ ID NO: 3+A98D,E,R,

SEQ ID NO: 3+S106A,W,

SEQ ID NO: 3+N117E,

SEQ ID NO: 3+H120V,D,K,N,

SEQ ID NO: 3+S124M,

SEQ ID NO: 3+P129D

SEQ ID NO: 3+E136Q,

SEQ ID NO: 3+S143W,

SEQ ID NO: 3+S161T,

SEQ ID NO: 3+S163A,G,

SEQ ID NO. 3+Y171L,

SEQ ID NO: 3+A172S,

SEQ ID NO: 3+N185Q,

SEQ ID NO: 3+V199M,

SEQ ID NO: 3+Y209W,

SEQ ID NO: 3+M222Q,

SEQ ID NO: 3+N238H,

SEQ ID NO: 3+V244T,

SEQ ID NO: 3+N261T,

SEQ ID NO: 3+L262N,Q,D,E

SEQ ID NO: 3+N76D+S163G+N238E

SEQ ID NO: 3+S156D+L262E

SEQ ID NO: 3+N238E+L262E

SEQ ID NO: 3+S3T+N76D+S156D+Y209W

SEQ ID NO: 3+H120D+S163G+N261D

SEQ ID NO: 3+S163G+N128Q+N238E+L262E

SEQ ID NO: 3+K27Q+H120D+S163G+N261D

SEQ ID NO: 3+V104T+H120D+S156D+L262E

SEQ ID NO: 3+G195E+V199M

SEQ ID NO: 3+S3T+V4I+N261D

SEQ ID NO: 3+A194P+G195E+V199M+V205I

SEQ ID NO: 3+H120D+A228V

SEQ ID NO: 3+S3T+V4I+A228V

SEQ ID NO: 3+H120D+N261D

SEQ ID NO: 3+H120D+S163G+N261D

SEQ ID NO: 3+N76D+A228V+L262E

SEQ ID NO: 3+N76D+Q137H+S141H+R145H+S163G+N238E

SEQ ID NO: 3+Q137H+S141H+R145H+N238E+L262E

SEQ ID NO: 3+S3T+N76D+Q137H+S141H+R145H+S156D+Y209W

SEQ ID NO: 3+H120D+Q137H+S141H+R145H+S163G+N261D

SEQ ID NO: 3+N76D+Q137H+5141H+R145H+A228V+N261D

SEQ ID NO: 3+A194P+G195E+V199M+V205I+A228V+N261D

SEQ ID NO: 3+N62D+H120D

SEQ ID NO: 3+H120D+N261D

SEQ ID NO: 3+N76D+N261D

SEQ ID NO: 3+N76D+A228V+N261D

SEQ ID NO: 3+A194P+G195E+V205I+N261D

SEQ ID NO: 3+N76D+H120D+N261D

SEQ ID NO: 3+H120D+S163G+N261D

SEQ ID NO: 3+S3T+Q59D+N76D

SEQ ID NO: 3+S3T+N76D+H120D

SEQ ID NO: 3+S3T+N76D+A194P+G195E+V199M+V205I

SEQ ID NO: 3+S3T+N76D+S156D

SEQ ID NO: 3+S3T+N76D+Y209W+N261D

SEQ ID NO: 3+S3T+N76D+H120D+Y209W

SEQ ID NO: 3+S3T+N76D+S156D+Y209W

SEQ ID NO: 3+S3T+V4I+N76D+A228V+N261D

SEQ ID NO: 3+S3T+V4I+N76D+H120D

SEQ ID NO: 3+H120D+P131F+A194P+N261D

SEQ ID NO: 3+N76D+E136H+A228V+N261D

SEQ ID NO: 3+N76D+N218S+A228V+N261D

SEQ ID NO: 3+N76D+N218Q+A228V+N261D

SEQ ID NO: 3+N76D+N218A+A228V+N261D

SEQ ID NO: 3+K27Q+R45E

SEQ ID NO: 3+N76D+A228V+L262E

SEQ ID NO: 3+R45E+A88S

SEQ ID NO: 3+S87E+K237E

SEQ ID NO: 3+N261D+L262E

SEQ ID NO: 3+S87E+L262E

SEQ ID NO: 3+S87E+N238E

SEQ ID NO: 3+K27Q+S87E

SEQ ID NO: 3+N76D+N117E

SEQ ID NO: 3+H120D+N238E

SEQ ID NO: 3+Q59D+L262E

SEQ ID NO: 3+K27Q+L262E

SEQ ID NO: 3+H120D+L262E

SEQ ID NO: 3+K27Q+Q59D

SEQ ID NO: 3+K27Q+S156D

SEQ ID NO: 3+K27Q+G61D

SEQ ID NO: 3+Q59D+N261D

SEQ ID NO: 3+Q59D+N117E

SEQ ID NO: 3+K237E+N261D

SEQ ID NO: 3+Q59D+N238E

SEQ ID NO: 3+A15T+H120D+N261D

SEQ ID NO: 3+N76D+S163G+N238E

SEQ ID NO: 3+H120D+S163G+L262E

SEQ ID NO: 3+H120D+S163G+N261D

SEQ ID NO: 3+Q59D+H120D

SEQ ID NO: 3+G61D+N76D

SEQ ID NO: 3+S3T+N76D

SEQ ID NO: 3+S3T+H120D

SEQ ID NO: 3+G61D+H120D

SEQ ID NO: 3+P55S+H120D

SEQ ID NO: 3+S163G+A228V

SEQ ID NO: 3+S163G+N261D

SEQ ID NO: 3+S3T+S163G

SEQ ID NO: 3+G61D+S163G

SEQ ID NO: 3+S156D+S163G

SEQ ID NO: 3+Q59D+S163G

SEQ ID NO: 3+N76D+S163G

SEQ ID NO: 3+P55S+S163G

SEQ ID NO: 3+H120D+S163G

SEQ ID NO: 3+T58L+Q59D

SEQ ID NO: 3+P55S+T58L

SEQ ID NO: 3+T58L+G97D

SEQ ID NO: 3+T58L+S106A

SEQ ID NO: 3+T58L+A228V

SEQ ID NO: 3+S3T+T58L

SEQ ID NO: 3+T58L+S156D

SEQ ID NO: 3+T58L+Y91H

SEQ ID NO: 3+T58L+H120D

SEQ ID NO: 3+T58L+S163G

SEQ ID NO: 3+S163G+N261D

SEQ ID NO: 3+T58L+N261D

SEQ ID NO: 3+T58L+N76D

SEQ ID NO: 3+S3T+N76D+H120D

SEQ ID NO: 3+S3T+N76D+A228V

SEQ ID NO: 3+S3T+N76D+S156D

SEQ ID NO: 3+S3T+N76D+Y209W

SEQ ID NO: 3+S3T+N76D+Y209W+V244T

SEQ ID NO: 3+N76D+H120D

SEQ ID NO: 3+N76D+S156D

SEQ ID NO: 3+H120D+S156D

SEQ ID NO 3+R45E+L262E

SEQ ID NO 3+Q59D+G61D

SEQ ID NO 3+S87E+L262E

SEQ ID NO 3+G61D+L262E

SEQ ID NO 3+Q59D+L262E

SEQ ID NO 3+R45E+Q59D

SEQ ID NO 3+Q59D+S156D

SEQ ID NO 3+S156D+L262E

SEQ ID NO 3+S163G+N238E+L262E

SEQ ID NO 3+S3T+V4I+S163G+N261D

SEQ ID NO 3+H120D+S163G+N261D

SEQ ID NO 3+Y91H+N117H+N238H

SEQ ID NO 3+T58L+S163G+N261D

SEQ ID NO 3+S3T+V4I+S163G+N261D

SEQ ID NO 3+S87E+S163G+L262E

SEQ ID NO 3+S156D+S163G+L262E

SEQ ID NO 3+T58LS163G+N261D

SEQ ID NO 3+S156DS163G+L262E

SEQ ID NO 3+S3T+N76D+Y209W+N261D+L262E

The subtilase variants of the invention preferably have improvedstability in liquid detergent compared with the parent protease,preferably the subtilase variants of the invention have improvedstability in liquid detergent compared with the protease having SEQ IDNO: 2 or SEQ ID NO: 3.

As examples of preferred subtilase variants of the invention haveimproved stability in liquid detergent and/or improved wash performancecompared with the parent enzyme can be mentioned:

SEQ ID NO: 3+R45E,D,Q

SEQ ID NO: 3+Q58L

SEQ ID NO: 3+Q59D,

SEQ ID NO: 3+G61D,

SEQ ID NO: 3+S87E,

SEQ ID NO: 3+G97S,

SEQ ID NO: 3+A98E,

SEQ ID NO: 3+N117E,

SEQ ID NO: 3+H120D,K,V

SEQ ID NO: 3+P129D

SEQ ID NO: 3+E136Q,

SEQ ID NO: 3+Q137H,

SEQ ID NO: 3+S156D,

SEQ ID NO: 3+S160A,

SEQ ID NO: 3+S163A,G,

SEQ ID NO: 3+V199M

SEQ ID NO: 3+M222Q

SEQ ID NO: 3+N261T or

SEQ ID NO: 3+L262EQ,N.

These preferred subtilase variants of the invention have improvedstability such as detergent stability and/or improved or on par washperformance compared with the parent subtilase. In this connectionimproved wash performance is intended to mean that the wash performanceof the variant is higher on at least one stain than the wash performanceof the parent subtilase where wash performance is determined using asuitable wash performance assay in a given detergent composition undersuitable conditions.

In one preferred embodiment, improved wash performance is measured usingAMSA test described in the Methods and Materials section of thisapplication.

The wash performance of the variant is preferably at least 1 unit higherthan the wash performance of the parent subtilase, preferably at least 2units higher, such as at least 3 units higher, such as at least 4 unitshigher, such as at least 5 units higher, such as at least 6 unitshigher, such as at least 7 units higher such as at least 8 units higher,such as at least 9 units higher.

The subtilase variants may further comprise one or more additionalalterations at one or more (e.g., several) other positions, selectedfrom the group consisting of positions: 3, 4, 9, 12, 14, 15, 40, 43, 68,72, 79, 86, 88, 92, 98, 99, 101, 120, 146, 183, 184, 188, 194, 216, 218,224, 228, 236, 245, 255, 261, 267 and 270, preferably positions 9, 15,68 and/or 120 (numbering according to SEQ ID NO: 1). It will be clear tothe skilled artisan that if a position has already been altered once,then it will not be altered a second time. In a preferred embodiment,the alteration at any of the positions selected from the groupconsisting of 3, 4, 9, 12, 14, 15, 40, 43, 68, 72, 79, 86, 88, 92, 98,99, 101, 120, 146, 183, 184, 188, 194, 216, 218, 224, 228, 236, 245,255, 261, 267 and 270 is a substitution. In a more preferred embodiment,the subtilase variant further comprises one or more substitutionsselected from the group consisting of 3{D, E, L}, 4I, 9 {H, K, R, G},12{D, E}, 14T, 15{G, M, S, T}, 40{A, G, M, S, T}, 43{D, E}, 63G, 68{A,G, I, L, M, S, T}, 72{V, L}, N76{D, E}, 79T, 86H, 88V, 92S, 98T, 99{E,T, A, G, M, D}, 101L, 120 {I, N}, 146S, 183{E, D}, 184{E, D}, 188G,194P, 216{D, E}, 218{E, D}, 224{S, A, T, G, M}, 228T, 236D, 245{H, K,R}, 255{D, E}, 261{E}, 267{I, L, V} and/or 270{G, M, S, T} (numberingaccording to SEQ ID NO: 1). In an even more preferred embodiment, thesubtilase variant further comprises one or more substitutions selectedfrom the group consisting of 53{D, E, L}, V4I, S9{H, K, R, G}, Q12{D,E}, P14T, A15{G, M, S, T}, P40{A, G, M, S, T}, N43{D, E}, V68{A, G, 1,L, M, S, T}, 172{V, L}, N76{D, E}, 179T, P86H, A88V, A92S, A98T, 599{E,T, A, G, M, D}, S101L, H120{I, N}, G146S, N183{E, D}, N184{E, D}, S188G,A194P, 5216{D, E}, N218{E, D}, T224{S, A, T, G, M}, A228T, S236D,Q245{H, K, R}, T255{D, E}, N261{, E}, L267{I, L, V} and/or A270{G, M, S,T} in the mature polypeptide of SEQ ID NO: 3 or a polypeptide having atleast 60%, preferably at least 70%, preferably at least 80%, preferablyat least 90%, preferably at least 95% sequence identity hereto, whereineach position corresponds to the corresponding position of the maturepolypeptide of SEQ ID NO: 1.

One embodiment further relates to a method for producing a subtilasevariant having improved stability and/or improved wash performancecompared to the subtilase having the amino acid sequence of SEQ ID NO: 2and/or SEQ ID NO 3, the method comprising the steps of

-   -   a) Substituting the amino acid in a position corresponding to        position 101 of SEQ ID NO: 1 with glutamic acid residue (E) in a        subtilase having at least 90% sequence identity to SEQ ID NO: 2,    -   b) Further introducing any of the following substitutions        position S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L,        Q59D,M,N,T, G61D,R, S87E, G97S, A98D,E,R, S106A,W, N117E,        H120V,D,K,N, S124M, P129D, E136Q, S143W, S161T, S163A,G, Y171L,        A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D or        L262N,Q,D,    -   c) Recovering the variant.

According to one embodiment and/or according to any of the embodimentsabove, the invention relates to a subtilase variant having at least 90%sequence identity to SEQ ID NO: 3, wherein the variant has a glutamicacid residue (E) in position corresponding to position 101 of SEQ ID NO:1, and where the variant has reduced cellulose binding compared to thesubtilase having the amino acid sequence of SEQ ID NO: 3. One embodimentrelates to a subtilase variant having at least 90% sequence identity toSEQ ID NO: 3, wherein the variant has a glutamic acid residue (E) inposition corresponding to position 101 of SEQ ID NO: 1, and where thevariant has reduced cellulose binding compared to the subtilase havingthe amino acid sequence of SEQ ID NO: 3 and wherein the variantcomprises a substitution of a positively charged amino acid residue onthe surface of the protease with a neutral or negatively chargedresidue; or a neutral residue on the surface of the protease issubstituted with a negatively charged residue. According to oneembodiment or any of the above embodiments the invention relates to asubtilase variant having at least 90% sequence identity to SEQ ID NO: 3,wherein the variant has a glutamic acid residue (E) in positioncorresponding to position 101 of SEQ ID NO: 1, and where the variant hasreduced cellulose binding compared to the subtilase having the aminoacid sequence of SEQ ID NO: 3 and/or wherein the variant comprises asubstitution of a positively charged amino acid residue on the surfaceof the protease with a neutral or negatively charged residue; or aneutral residue on the surface of the protease is substituted with anegatively charged residue wherein the subtilase variant comprising asubstitution selected from the group consisting of V4D,E,I,R10N,Q,D,E,S, H17D, K27S,N,Q,E,D, N43E, 144V, R45E,D,Q,N, G46D, S49N,D,P52E, G53D,E, Q59D, G61D, N62D, L75D, N76D, 179D, S87E, G97D, A98E,*103aE, I104T, N117E, H120D, E136K,Q, S156D, R170E,Q,N,D,S N185D, G195E,N218A, K235L,W,N,Q,E,S, K237N,Q,D,E,S, N238D,E, V244D, R246Q,E,D,R247S,E, Q, D, K251S,D,Q,E,N, N261D, L262D,E and S265H, preferable thesubstitutions are selected among N117E, S156D, N238E, N261D and L262Eand preferably the variant further comprise a substitution selectedamong: S3T, N128Q, Q137H, S141H, R145H, S163G, A194P, V199M, V205I ,N218Q or A228V. According to one embodiment and/or according to any ofthe above embodiment the invention relates to a subtilase variant havingat least 90% sequence identity to SEQ ID NO: 3, wherein the variant hasa glutamic acid residue (E) in position corresponding to position 101 ofSEQ ID NO: 1, where the variant has reduced cellulose binding comparedto the subtilase having the amino acid sequence of SEQ ID NO: 3 andwherein the subtilase variant further comprising a substitutionsselected among N117E+S3T, S156D+S3T, N238E+S3T, N261D+S3T, L262E+S3T,N117E+N128Q, S156D+N128Q, N238E+N128Q, N261D+N128Q, L262E+N128Q,N117E+Q137H, S156D+Q137H, N238E+Q137H, N261D+Q137H, L262E+Q137H,N117E+S141H, S156D+S141H, N238E+S141H, N261D+S141H, L262E+S141H,N117E+R145H, S156D+R145H, N238E+R145H, N261D+R145H, L262E+R145H,N117E+S163G, S156D+S163G, N238E+S163G, N261D+S163G, L262E+S163G,N117E+A194P, S156D+A194P, N238E+A194P, N261D+A194P, L262E+A194P,N117E+V199M, S156D+V199M, N238E+V199M, N261D+V199M, L262E+V199M,N117E+V205I , S156D+V205I , N238E+V205I, N261D+V205I, L262E+V205I ,N117E+N218Q, S156D+N218Q, N238E+N218Q, N261D+N218Q, L262E+N218Q,N117E+A228V,S156D+A228V, N238E+A228V, N261D+A228V, L262E+A228V,S156D+N262E. According to one embodiment and/or according to any of theabove embodiment the invention relates to a subtilase variant having atleast 90% sequence identity to SEQ ID NO: 3, wherein the variant has aglutamic acid residue (E) in position corresponding to position 101 ofSEQ ID NO: 1, where the variant has reduced cellulose binding comparedto the subtilase having the amino acid sequence of SEQ ID NO: 3, andwherein the subtilase variant further comprising a substitutionsselected among:

SEQ ID NO: 3+V4D,E,I

SEQ ID NO: 3+R10N,Q,D,E,S,

SEQ ID NO: 3+H17D,

SEQ ID NO: 3+K27S,N,Q,E,D,

SEQ ID NO: 3+R45E,D,Q,N,

SEQ ID NO: 3+G53D,

SEQ ID NO: 3+Q59D,

SEQ ID NO: 3+G61D,

SEQ ID NO: 3+L75D,

SEQ ID NO: 3+N76D,

SEQ ID NO: 3+I79D,

SEQ ID NO: 3+S87E,

SEQ ID NO: 3+G97D,

SEQ ID NO: 3+A98E,

SEQ ID NO: 3+*103aE,

SEQ ID NO:3+N117E,

SEQ ID NO:3+H120D,

SEQ ID NO:3+E136K,Q,

SEQ ID NO. 3+S156D,

SEQ ID NO: 3+R170E,Q,N,D,

SEQ ID NO: 3+N185D,

SEQ ID NO: 3+G195E,

SEQ ID NO: 3+K235L,W,N,Q,E,S,

SEQ ID NO: 3+K237N,Q,D,E,S,

SEQ ID NO: 3+N238D,E,

SEQ ID NO: 3+V244D

SEQ ID NO: 3+R246Q,E,D,

SEQ ID NO: 3+R247S,E,

SEQ ID NO: 3+K251S,D,Q,E,N,

SEQ ID NO: 3+N261D,

SEQ ID NO: 3+L262D,E

SEQ ID NO: 3+S265H

SEQ ID NO: 3+A194P+G195E

SEQ ID NO: 3+G195E+V199M

SEQ ID NO: 3+N76D+A228V+N261D;

SEQ ID NO: 3+N76D+S163G+N238E

SEQ ID NO: 3+S156D+L262E

SEQ ID NO: 3+N238E+L262E

SEQ ID NO: 3+S3T+N76D+S156D+Y209W

SEQ ID NO: 3+K27Q+H120D+S163G+N261D

SEQ ID NO: 3+V104T+H120D+S156D+L262E

SEQ ID NO: 3+V104T+S156D+L262E

SEQ ID NO: 3+Q137H+S141H+R145H+N238E+L262E

SEQ ID NO: 3+S3T+V4I+A228V;

SEQ ID NO: 3+H120D S163G N261D

SEQ ID NO: 3+N76D+5101E+A228V+L262E;

SEQ ID NO: 3+N76D+Q137H+S141H+R145H+S163G+N238E

SEQ ID NO: 3+S3T+N76D+Q137H+S141H+R145H+S156D+Y209W

SEQ ID NO: 3+H120D+Q137H+S141H+R145H+S163G+N261D

SEQ ID NO: 3+A194P+G195E+V199M+V205I ;

SEQ ID NO: 3+S3T+N76D+A194P+G195E+V199M+V205I ;

SEQ ID NO: 3+A228V+N261D;

SEQ ID NO: 3+N76D+A228V;

SEQ ID NO: 3+S3T+V4I+N261D;

SEQ ID NO: 3+H120D+A228V;

SEQ ID NO: 3+N76D+N261D;

SEQ ID NO: 3+A194P+G195E+V199M+V205I+A228V+N261D;

SEQ ID NO: 3+A194P+G195E+V205I+A228V; or

SEQ ID NO: 3+H120D+N261D.

Preferably the variants are selected from the group consisting of:

SEQ ID NO: 3+N238E+L262E

SEQ ID NO: 3+S156D+L262E

SEQ ID NO: 3+S3T+V4I+A228V;

SEQ ID NO: 3+G195E+V199M

SEQ ID NO: 3+H120D S163G N261D

SEQ ID NO: 3+N76D+A228V+N261D;

SEQ ID NO: 3+S3T+N76D+S156D+Y209W

SEQ ID NO: 3+Q137H+S141H+R145H+N238E+L262E

SEQ ID NO: 3+Q137H+S141H+R145H+S156D+L262E

SEQ ID NO: 3+N76D+Q137H+S141H+R145H+A228V+N261D;

SEQ ID NO: 3+N76D+Q137H+S141H+R145H+S163G+N238E

SEQ ID NO: 3+H120D+Q137H+S141H+R145H+S163G+N261D

SEQ ID NO: 3+S3T+N76D+Q137H+S141H+R145H+S156D+Y209W,

wherein the positions correspond to the positions in SEQ ID NO: 1 andwherein the subtilase variant has at least at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95%, such as at least 98% or such as at least 99% sequence identity toSEQ ID NO: 3. One embodiment relates to a nucleotide sequence encoding avariant according to any of the above embodiments, an expression vectorcomprising the nucleotide sequence, a recombinant host cell comprisingthe nucleotide sequence or the expression vector. One embodiment furtherrelates to a method for producing a subtilase variant having reducedcellulose binding compared to the subtilase having the amino acidsequence of SEQ ID NO: 2, the method comprising the steps of

-   -   a) Substituting the amino acid in a position corresponding to        position 101 of SEQ ID NO: 1 with glutamic acid residue (E) in a        subtilase having at least 90% sequence identity to SEQ ID NO: 2,    -   b) Further introducing any of the following substitutions        position 4D,E,I, 10N,Q,D,E,S, H17D, K27S,N,Q,E,D, N43E, 144V,        R45E,D,Q,N, G46D, S49N,D, P52E, G53D,E, Q59D, G61D, N62D, L75D,        N76D, 179D, S87E, G97D, A98E, *103aE, I104T, N117E, H120D,        E136K,Q, S156D, R170E,Q,N,D,S N185D, G195E, N218A,        K235L,W,N,Q,E,S, K237N,Q,D,E,S, N238D,E, V244D, R246Q,E,D,        R247S,E, Q, D, K251S,D,Q,E,N, N261D, L262D,E and S265H;    -   c) Recovering the variant.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R.L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis. In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for protease activity to identifyamino acid residues that are critical to the activity of the molecule.The active site of the enzyme or other biological interaction can alsobe determined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. For BPN′ (SEQ ID NO: 1) the catalytictriad comprising the amino acids S221, H64, and D32 is essential forprotease activity of the enzyme.

The subtilase variants may consist of 150 to 350, e.g., 175 to 330, 200to 310, 220 to 300, 240 to 290, 260 to 280 or 269, 270, 271, 272, 273,274 or 275 amino acids.

In one embodiment, the subtilase variant has improved wash performance.In another embodiment, the subtilase variant has improved stability,preferably improved stability during wash.

In an embodiment, the subtilase variant has improved stability in liquiddetergent compared to the parent enzyme wherein the stability ismeasured using the ‘stability assay’ as described in example 4 in theMaterials and Methods section herein. In an embodiment, the subtilasevariant has improved stability compared to the polypeptide of SEQ ID NO:2, wherein stability is measured using the ‘stability assay’ asdescribed in example 4 in the Materials and Methods section herein. Inan embodiment, the subtilase variant has improved stability compared tothe polypeptide of SEQ ID NO: 3, wherein stability is measured using the‘stability assay’ as described in example 4 in the Materials and Methodssection herein.

In an embodiment, the subtilase variant has improved wash performancecompared to the parent enzyme wherein wash performance is measured usingthe Automatic Mechanical Stress Assay (AMSA) as described in example 7in the Materials and Methods section herein. In an embodiment, thesubtilase variant has improved wash performance compared to thepolypeptide of SEQ ID NO: 2 wherein wash performance is measured usingthe Automatic Mechanical Stress Assay (AMSA) as described in example 7in the Materials and Methods section herein. In an embodiment, thesubtilase variant has improved wash performance compared to thepolypeptide of SEQ ID NO: 3, wherein wash performance is measured usingthe Automatic Mechanical Stress Assay (AMSA) as described in example 7in the Materials and Methods section herein.

Parent Protease

Enzymes cleaving the amide linkages in protein substrates are classifiedas proteases, or (interchangeably) peptidases.

Serine Proteases

A serine protease is an enzyme, which catalyzes the hydrolysis ofpeptide bonds, and in which there is an essential serine residue at theactive site.

The bacterial serine proteases have molecular weights in the 20,000 to45,000 Dalton range. They are inhibited by diisopropylfluorophosphate.They hydrolyze simple terminal esters and are similar in activity toeukaryotic chymotrypsin, also a serine protease. A more narrow term,alkaline protease, covering a sub-group, reflects the high pH optimum ofsome of the serine proteases, from pH 9.0 to 11.0.

Subtilases

A sub-group of the serine proteases tentatively designated subtilaseshas been proposed by Siezen et al. (1991), Protein Eng. 4:719-737 andSiezen et al. (1997), Protein Science 6:501-523. They are defined byhomology analysis of more than 170 amino acid sequences of serineproteases previously referred to as subtilisin-like proteases. Asubtilisin was previously often defined as a serine protease produced byGram-positive bacteria or fungi, and according to Siezen et al. now is asubgroup of the subtilases. A wide variety of subtilases have beenidentified, and the amino acid sequence of a number of subtilases hasbeen determined. For a more detailed description of such subtilases andtheir amino acid sequences reference is made to Siezen et al. (1997).

Subtilisins

A subgroup of the subtilases is the subtilisins which are serineproteases from the family S8, in particular from the subfamily SBA, asdefined by the MEROPS database(http://merops.sangerac.uk/cgi-bin/famsum?family=S8).

BPN′ and Savinase have the MEROPS numbers S08.034 and S08.003,respectively.

Parent Subtilase

The term “parent subtilase” describes a subtilase defined according toSiezen et al. (1997), Protein Science 6:501-523. For further details seedescription of “Subtilases” above. A parent subtilase may also be asubtilase isolated from a natural source, wherein subsequentmodifications (such as replacement(s) of the amino acid side chain(s),substitution(s), deletion(s) and/or insertion(s)) have been made whileretaining the characteristic of a subtilase. Furthermore, a parentsubtilase may be a subtilase which has been prepared by the DNAshuffling technique.

Alternatively, the term “parent subtilase” may be termed “wild typesubtilase”. The parent subtilase is preferably of the subtilisinsubgroups. One subgroup of the subtilases, I-S1 or “true” subtilisins,comprises the “classical” subtilisins, such as subtilisin 168 (BSS168),subtilisin BPN′, subtilisin Carlsberg (ALCALASE®, NOVOZYMES A/S), andsubtilisin DY (BSSDY).

A further subgroup of the subtilases, I-S2 or high alkaline subtilisins,is recognized by Siezen et al. (supra). Sub-group I-S2 proteases aredescribed as highly alkaline subtilisins and comprises enzymes such assubtilisin PB92 (BAALKP) (MAXACAL®, Genencor International Inc.),subtilisin 309 (SAVINASE®, NOVOZYMES A/S), subtilisin 147 (BLS147)(ESPERASE®, NOVOZYMES A/S), and alkaline elastase YaB (BSEYAB). BPN′ issubtilisin BPN′ from B. amyloliquefaciens BPN′ has the amino acidsequence SEQ ID NO: 1.

For reference, table 1 below gives a list of some acronyms for varioussubtilases mentioned herein. For further acronyms, see Siezen et al.(1991 and 1997).

TABLE 1 Acronyms of various subtilases Organism Enzyme Acronym Bacillussubtilis 168 subtilisin I168, apr BSS168 Bacillus amyloliquefacienssubtilisin BPN' (NOVO) BASBPN Bacillus subtilis DY subtilisin DY BSSDYBacillus licheniformis subtilisin Carlsberg BLSCAR Bacillus lentussubtilisin 309 BLSAVI Bacillus lentus subtilisin 147 BLS147 Bacillusalcalophilus PB92 subtilisin PB92 BAPB92 Bacillus YaB alkaline elastaseYaB BYSYAB Bacillus sp. NKS-21 subtilisin ALP I BSAPRQ Bacillus sp.G-825-6 subtilisin Sendai BSAPRS Thermoactinomyces vulgaris ThermitaseTVTH ER

Homologous Subtilase Sequences

The homology between two amino acid sequences is in this contextdescribed by the parameter “identity” for purposes of the presentinvention, the degree of identity between two amino acid sequences isdetermined using the Needleman-Wunsch algorithm as described above. Theoutput from the routine is besides the amino acid alignment thecalculation of the “Percent Identity” between the two sequences.

Based on this description, it is routine for a person skilled in the artto identify suitable homologous subtilases, which can be modifiedaccording to the invention.

Substantially homologous parent subtilisin variants may have one or more(several) amino acid substitutions, deletions and/or insertions, in thepresent context the term “one or more” is used interchangeably with theterm “several”. These changes are preferably of a minor nature, that isconservative amino acid substitutions as described above and othersubstitutions that do not significantly affect the three-dimensionalfolding or activity of the protein or polypeptide; small deletions,typically of one to about 30 amino acids; and small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue, a small linker peptide of up to about 20-25 residues, or asmall extension that facilitates purification (an affinity tag), such asa poly-histidine tract, or protein.

Although the changes described above preferably are of a minor nature,such changes may also be of a substantive nature such as fusion oflarger polypeptides of up to 300 amino acids or more both as amino- orcarboxyl-terminal extensions.

The polypeptide of SEQ ID NO: 3 or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encoding a parentfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵5, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally.

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial protease. For example, the parent may be aGram-positive bacterial polypeptide such as a Bacillus, Clostridium,Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,Staphylococcus, Streptococcus, or Streptomyces protease, or aGram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma protease.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis protease

In one aspect, the parent is a Bacillus amyloliquefaciens protease,e.g., the protease of SEQ ID NO: 1 or the mature polypeptide thereof.

In another aspect, the parent is a Bacillus lentus protease, e.g., theprotease of SEQ ID NO: 2 or the mature polypeptide thereof.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes.

Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding a parentmay then be obtained by similarly screening a genomic DNA or cDNAlibrary of another microorganism or mixed DNA sample. Once apolynucleotide encoding a parent has been detected with the probe(s),the polynucleotide can be isolated or cloned by utilizing techniquesthat are known to those of ordinary skill in the art.

Preparation of Variants

The present invention also relates to methods for obtaining a subtilasevariant having protease activity.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology and similar technologies whereinoligonucleotides are synthesized and assembled upon photo-programmablemicrofluidic chips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure. Other methods that can be used include error-prone PCR, phagedisplay and region-directed mutagenesis.

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells. Mutagenized DNA molecules thatencode active polypeptides can be recovered from the host cells andrapidly sequenced using standard methods in the art. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to polynucleotides encoding a variantof the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis crylllA gene, E. coli lac operon, E. coli trcpromoter, Streptomyces coelicolor agarase gene (dagA), and prokaryoticbeta-lactamase gene, as well as the tac promoter

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis crylllA gene and a Bacillus subtilis SP82 gene.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase, Rhizomucor miehei asparticproteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art.

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation, competent cell transformation electroporationor conjugation. The introduction of DNA into an E. coli cell may beeffected by protoplast transformation or electroporation. Theintroduction of DNA into a Streptomyces cell may be effected byprotoplast transformation, electroporation, conjugation, ortransduction. The introduction of DNA into a Pseudomonas cell may beeffected by electroporation, or conjugation. The introduction of DNAinto a Streptococcus cell may be effected by natural competence,protoplast transformation, electroporation or conjugation. However, anymethod known in the art for introducing DNA into a host cell can beused.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants with protease activity. These detectionmethods include, but are not limited to, use of specific antibodies,formation of an enzyme product, or disappearance of an enzyme substrate.For example, an enzyme assay may be used to determine the activity ofthe variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction to obtain substantially pure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Compositions

In one certain aspect, the subtilase variants according to the inventionhave improved wash performance compared to the parent enzyme or comparedto a protease having the identical amino acid sequence of said variantbut not having the alterations at one or more of said specifiedpositions or compared to the polypeptide of SEQ ID NO: 2 or compared tothe polypeptide of SEQ ID NO 3, wherein wash performance is measuredusing the Automatic Mechanical Stress Assay (AMSA).

In another certain aspect, the subtilase variants according to theinvention have improved stability, preferably improved stability duringwash, compared to the parent enzyme or compared to a protease having theidentical amino acid sequence of said variant but not having thealterations at one or more of said specified positions or compared tothe polypeptide of SEQ ID NO: 2 or compared to the polypeptide of SEQ IDNO 3, wherein stability is measured using the ‘stability assay’ asdescribed in example 4 in the Materials and Methods section herein.

Compositions may be detergent compositions comprising subtilase variantsaccording to the invention which may be used in a cleaning process suchas laundry or hard surface cleaning.

The choice of additional components is within the skill of the artisanand includes conventional ingredients, including the exemplarynon-limiting components set forth below. The choice of components mayinclude, for fabric care, the consideration of the type of fabric to becleaned, the type and/or degree of soiling, the temperature at whichcleaning is to take place, and the formulation of the detergent product.Although components mentioned below are categorized by general headeraccording to a particular functionality, this is not to be construed asa limitation, as a component may comprise additional functionalities aswill be appreciated by the skilled artisan.

Enzyme of the Present Invention

In one embodiment of the present invention, the a polypeptide of thepresent invention may be added to a detergent composition in an amountcorresponding to 0.01-200 mg of enzyme protein per liter of wash liqour,preferably 0.05-50 mg of enzyme protein per liter of wash liqour, inparticular 0.1-10 mg of enzyme protein per liter of wash liqour.

A composition for use in automatic dishwash (ADW), for example, mayinclude 0.0001%-50%, such as 0.001%-30%, such as 0.01%-20%, such as0.5-15% of enzyme protein by weight of the composition.

A composition for use in laundry granulation, for example, may include0.0001%-50%, such as 0.001%-20%, such as 0.01%-10%, such as 0.05%-5% ofenzyme protein by weight of the composition.

A composition for use in laundry liquid, for example, may include0.0001%-10%, such as 0.001-7%, such as 0.1%-5% of enzyme protein byweight of the composition.

The enzyme(s) in a detergent composition may be stabilized usingconventional stabilizing agents, e.g., a polyol such as propylene glycolor glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or aboric acid derivative, e.g., an aromatic borate ester, or a phenylboronic acid derivative such as 4-formylphenyl boronic acid, and thecomposition may be formulated as described in, for example, WO 92/19709and WO 92/19708 or the subtilase variants according to the invention maybe stabilized using peptide aldehydes or ketones such as described in WO2005/105826 and WO 2009/118375.

A variant of the present invention may also be incorporated in thedetergent formulations disclosed in WO 97/07202, which is herebyincorporated by reference.

Surfactants

The detergent composition may comprise one or more surfactants, whichmay be anionic and/or cationic and/or non-ionic and/or semi-polar and/orzwitterionic, or a mixture thereof. In a particular embodiment, thedetergent composition includes a mixture of one or more nonionicsurfactants and one or more anionic surfactants. The surfactant(s) istypically present at a level of from about 0.1% to 60% by weight, suchas about 1% to about 40%, or about 3% to about 20%, or about 3% to about10%. The surfactant(s) is chosen based on the desired cleaningapplication, and includes any conventional surfactant(s) known in theart. Any surfactant known in the art for use in detergents may beutilized.

When included therein the detergent will usually contain from about 1%to about 40% by weight, such as from about 5% to about 30%, includingfrom about 5% to about 15%, or from about 20% to about 25% of an anionicsurfactant. Non-limiting examples of anionic surfactants includesulfates and sulfonates, in particular, linear alkylbenzenesulfonates(LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS),phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates,alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonatesand disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate(SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS),alcohol ethersulfates (AES or AEOS or FES, also known as alcoholethoxysulfates or fatty alcohol ether sulfates), secondaryalkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates,sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methylesters (alpha-SFMe or SES) including methyl ester sulfonate (MES),alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid(DTSA), fatty acid derivatives of amino acids, diesters and monoestersof sulfo-succinic acid or soap, and combinations thereof.

When included therein the detergent will usually contain from about 0%to about 10% by weight of a cationic surfactant. Non-limiting examplesof cationic surfactants include alklydimethylethanolamine quat (ADMEAQ),cetyltrimethylammonium bromide (CTAB), dimethyldistearylammoniumchloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternaryammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, andcombinations thereof.

When included therein the detergent will usually contain from about 0.2%to about 40% by weight of a non-ionic surfactant, for example from about0.5% to about 30%, in particular from about 1% to about 20%, from about3% to about 10%, such as from about 3% to about 5%, or from about 8% toabout 12%. Non-limiting examples of non-ionic surfactants includealcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylatedfatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such asethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenolethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides(APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fattyacid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides(EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine(glucamides, GA, or fatty acid glucamide, FAGA), as well as productsavailable under the trade names SPAN and TWEEN, and combinationsthereof.

When included therein the detergent will usually contain from about 0%to about 10% by weight of a semipolar surfactant. Non-limiting examplesof semipolar surfactants include amine oxides (AO) such asalkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide andN-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acidalkanolamides and ethoxylated fatty acid alkanolamides, and combinationsthereof.

When included therein the detergent will usually contain from about 0%to about 10% by weight of a zwitterionic surfactant. Non-limitingexamples of zwitterionic surfactants include betaine,alkyldimethylbetaine, sulfobetaine, and combinations thereof.

Hydrotropes

A hydrotrope is a compound that solubilises hydrophobic compounds inaqueous solutions (or oppositely, polar substances in a non-polarenvironment). Typically, hydrotropes have both hydrophilic and ahydrophobic character (so-called amphiphilic properties as known fromsurfactants); however the molecular structure of hydrotropes generallydo not favor spontaneous self-aggregation. Hydrotropes do not display acritical concentration above which self-aggregation occurs as found forsurfactants and lipids forming miceller, lamellar or other well definedmeso-phases. Instead, many hydrotropes show a continuous-typeaggregation process where the sizes of aggregates grow as concentrationincreases. However, many hydrotropes alter the phase behavior,stability, and colloidal properties of systems containing substances ofpolar and non-polar character, including mixtures of water, oil,surfactants, and polymers. Hydrotropes are classically used acrossindustries from pharma, personal care, food, to technical applications.Use of hydrotropes in detergent compositions allow for example moreconcentrated formulations of surfactants (as in the process ofcompacting liquid detergents by removing water) without inducingundesired phenomena such as phase separation or high viscosity.

The detergent may contain 0-5% by weight, such as about 0.5 to about 5%,or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in theart for use in detergents may be utilized. Non-limiting examples ofhydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate(STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS),sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers,sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodiumethylhexyl sulfate, and combinations thereof.

Builders and Co-Builders

The detergent composition may contain about 0-65% by weight, such asabout 5% to about 45% of a detergent builder or co-builder, or a mixturethereof. In a dish wash detergent, the level of builder is typically40-65%, particularly 50-65%. The builder and/or co-builder mayparticularly be a chelating agent that forms water-soluble complexeswith Ca and Mg. Any builder and/or co-builder known in the art for usein laundry detergents may be utilized. Non-limiting examples of buildersinclude zeolites, diphosphates (pyrophosphates), triphosphates such assodium triphosphate (STP or STPP), carbonates such as sodium carbonate,soluble silicates such as sodium metasilicate, layered silicates (e.g.,SKS-6 from Hoechst), ethanolamines such as 2-aminoethan-1-ol (MEA),diethanolamine (DEA, also known as iminodiethanol), triethanolamine(TEA, also known as 2,2′,2″-nitrilotriethanol), and carboxymethyl inulin(CMI), and combinations thereof.

The detergent composition may also contain 0-20% by weight, such asabout 5% to about 10%, of a detergent co-builder, or a mixture thereof.The detergent composition may include include a co-builder alone, or incombination with a builder, for example a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocarboxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid(HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA),diethylenetriaminepentakis(methylenephosphonic acid) (DTPMPA or DTMPA),N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoaceticacid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), asparticacid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid(SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL),N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid(MIDA), α-alanine-N, N-diacetic acid (α-ALDA), serine-N, N-diacetic acid(SEDA), isoserine-N, N-diacetic acid (ISDA), phenylalanine-N, N-diaceticacid (PHDA), anthranilic acid-N, N-diacetic acid (ANDA), sulfanilicacid-N, N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TUDA) andsulfomethyl-N, N-diacetic acid (SMDA),N-(2-hydroxyethyl)-ethylidenediamine-N, N′, N′-triacetate (HEDTA),diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonicacid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), andcombinations and salts thereof. Further exemplary builders and/orco-builders are described in, e.g., WO 09/102854, U.S. Pat. No.5,977,053

Bleaching Systems

The detergent may contain 0-50% by weight, such as about 0.1% to about25%, of a bleaching system. Any bleaching system known in the art foruse in laundry detergents may be utilized. Suitable bleaching systemcomponents include bleaching catalysts, photobleaches, bleachactivators, sources of hydrogen peroxide such as sodium percarbonate andsodium perborates, preformed peracids and mixtures thereof. Suitablepreformed peracids include, but are not limited to, peroxycarboxylicacids and salts, percarbonic acids and salts, perimidic acids and salts,peroxymonosulfuric acids and salts, for example, Oxone (R), and mixturesthereof. Non-limiting examples of bleaching systems includeperoxide-based bleaching systems, which may comprise, for example, aninorganic salt, including alkali metal salts such as sodium salts ofperborate (usually mono- or tetra-hydrate), percarbonate, persulfate,perphosphate, persilicate salts, in combination with a peracid-formingbleach activator. The term bleach activator is meant herein as acompound which reacts with peroxygen bleach like hydrogen peroxide toform a peracid. The peracid thus formed constitutes the activatedbleach. Suitable bleach activators to be used herein include thosebelonging to the class of esters amides, imides or anhydrides. Suitableexamples are tetracetylethylene diamine (TAED), sodium4-[(3,5,5-trimethyl hexanoyl)oxy]benzene sulfonate (ISONOBS), diperoxydodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate (LOBS),4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS),4-(nonanoyloxy)-benzenesulfonate (NOBS), and/or those disclosed inWO98/17767. A particular family of bleach activators of interest wasdisclosed in EP624154 and particularly preferred in that family isacetyl triethyl citrate (ATC). ATC or a short chain triglyceride liketriacetin has the advantage that it is environmental friendly as iteventually degrades into citric acid and alcohol. Furthermore acetyltriethyl citrate and triacetin has a good hydrolytical stability in theproduct upon storage and it is an efficient bleach activator. FinallyATC provides a good building capacity to the laundry additive.Alternatively, the bleaching system may comprise peroxyacids of, forexample, the amide, imide, or sulfone type. The bleaching system mayalso comprise peracids such as 6-(phthalimido)peroxyhexanoic acid (PAP).The bleaching system may also include a bleach catalyst. In someembodiments the bleach component may be an organic catalyst selectedfrom the group consisting of organic catalysts having the followingformulae:

(iii) and mixtures thereof; wherein each R¹ is independently a branchedalkyl group containing from 9 to 24 carbons or linear alkyl groupcontaining from 11 to 24 carbons, preferably each R¹ is independently abranched alkyl group containing from 9 to 18 carbons or linear alkylgroup containing from 11 to 18 carbons, more preferably each R¹ isindependently selected from the group consisting of 2-propylheptyl,2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl,n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl andiso-pentadecyl. Other exemplary bleaching systems are described, e.g. inWO2007/087258, WO2007/087244, WO2007/087259 and WO2007/087242. Suitablephotobleaches may for example be sulfonated zinc phthalocyanine

Polymers

The detergent may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2%or 0.2-1% of a polymer. Any polymer known in the art for use indetergents may be utilized. The polymer may function as a co-builder asmentioned above, or may provide antiredeposition, fiber protection, soilrelease, dye transfer inhibition, grease cleaning and/or anti-foamingproperties. Some polymers may have more than one of the above-mentionedproperties and/or more than one of the below-mentioned motifs. Exemplarypolymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol)(PVA), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) orpoly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine),carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA/PMA,poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers,hydrophobically modified CMC (HM-CMC) and silicones, copolymers ofterephthalic acid and oligomeric glycols, copolymers of poly(ethyleneterephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP,poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO)and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplarypolymers include sulfonated polycarboxylates, polyethylene oxide andpolypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Otherexemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of theabove-mentioned polymers are also contemplated.

Fabric Hueing Agents

The detergent compositions of the present invention may also includefabric hueing agents such as dyes or pigments, which when formulated indetergent compositions can deposit onto a fabric when said fabric iscontacted with a wash liquor comprising said detergent compositions andthus altering the tint of said fabric through absorption/reflection ofvisible light. Fluorescent whitening agents emit at least some visiblelight. In contrast, fabric hueing agents alter the tint of a surface asthey absorb at least a portion of the visible light spectrum. Suitablefabric hueing agents include dyes and dye-clay conjugates, and may alsoinclude pigments. Suitable dyes include small molecule dyes andpolymeric dyes. Suitable small molecule dyes include small molecule dyesselected from the group consisting of dyes falling into the Colour Index(C.I.) classifications of Direct Blue, Direct Red, Direct Violet, AcidBlue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, ormixtures thereof, for example as described in WO 2005/03274, WO2005/03275, WO 2005/03276 and EP 1876226 (hereby incorporated byreference). The detergent composition preferably comprises from about0.00003 wt % to about 0.2 wt %, from about 0.00008 wt % to about 0.05 wt%, or even from about 0.0001 wt % to about 0.04 wt % fabric hueingagent. The composition may comprise from 0.0001 wt % to 0.2 wt % fabrichueing agent, this may be especially preferred when the composition isin the form of a unit dose pouch. Suitable hueing agents are alsodisclosed in, e.g. WO 2007/087257 and WO 2007/087243.

Additional Enzymes

The detergent additive as well as the detergent composition may compriseone or more (additional) enzymes such as a protease, lipase, cutinase,an amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase,galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.

In general, the properties of the selected enzyme(s) should becompatible with the selected detergent, (i.e., pH-optimum, compatibilitywith other enzymatic and non-enzymatic ingredients, etc.), and theenzyme(s) should be present in effective amounts.

Cellulases

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593,5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Example of cellulases exhibiting endo-beta-1,4-glucanase activity (EC3.2.1.4) are those having described in WO 02/099091.

Other examples of cellulases include the family 45 cellulases describedin WO 96/29397, and especially variants thereof having substitution,insertion and/or deletion at one or more of the positions correspondingto the following positions in SEQ ID NO: 8 of WO 02/099091:2, 4, 7, 8,10, 13, 15, 19, 20, 21, 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43,44, 48, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82,84, 86, 88, 90, 91, 93, 95, 95d, 95h, 95j, 97, 100, 101, 102, 103, 113,114, 117, 119, 121, 133, 136, 137, 138, 139, 140a, 141, 143a, 145, 146,147, 150e, 150j, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160c,160e, 160k, 161, 162, 164, 165, 168, 170, 171, 172, 173, 175, 176, 178,181, 183, 184, 185, 186, 188, 191, 192, 195, 196, 200, and/or 20,preferably selected among P19A, G20K, Q44K, N48E, Q119H or Q146 R.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Proteases

Suitable additional proteases include those of bacterial, fungal, plant,viral or animal origin e.g. vegetable or microbial origin. Microbialorigin is preferred. Chemically modified or protein engineered mutantsare included. It may be an alkaline protease, such as a serine proteaseor a metalloprotease. A serine protease may for example be of the 51family, such as trypsin, or the S8 family such as subtilisin. Ametalloproteases protease may for example be a thermolysin from e.g.family M4 or other metalloprotease such as those from M5, M7 or M8families.

The term “subtilases” refers to a sub-group of serine protease accordingto Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al.Protein Science 6 (1997) 501-523. Serine proteases are a subgroup ofproteases characterized by having a serine in the active site, whichforms a covalent adduct with the substrate. The subtilases may bedivided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitasefamily, the Proteinase K family, the Lantibiotic peptidase family, theKexin family and the Pyrolysin family.

Examples of subtilases are those derived from Bacillus such as Bacilluslentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacilluspumilus and Bacillus gibsonii described in; U.S. Pat. No. 7,262,042 andWO 09/021867, and subtilisin lentus, subtilisin Novo, subtilisinCarlsberg, Bacillus licheniformis, subtilisin BPN′, subtilisin 309,subtilisin 147 and subtilisin 168 described in WO 89/06279 and proteasePD138 described in (WO 93/18140). Other useful proteases may be thosedescribed in WO 92/175177, WO 01/016285, WO 02/026024 and WO 02/016547.Examples of trypsin-like proteases are trypsin (e.g. of porcine orbovine origin) and the Fusarium protease described in WO 89/06270, WO94/25583 and WO 05/040372, and the chymotrypsin proteases derived fromCellumonas described in WO 05/052161 and WO 05/052146.

A further preferred protease is the alkaline protease from Bacilluslentus DSM 5483, as described for example in WO 95/23221, and variantsthereof which are described in WO 92/21760, WO 95/23221, EP 1921147 andEP 1921148.

Examples of metalloproteases are the neutral metalloprotease asdescribed in WO 07/044993 (Genencor Int.) such as those derived fromBacillus amyloliquefaciens.

Examples of useful proteases are the variants described in: WO 92/19729,WO 96/034946, WO 98/20115, WO 98/20116, WO 99/011768, WO 01/44452, WO03/006602, WO 04/03186, WO 04/041979, WO 07/006305, WO 11/036263, WO11/036264, especially the variants with substitutions in one or more ofthe following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130,160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235,236, 245, 248, 252 and 274 using the BPN′ numbering. More preferred thesubtilase variants may comprise the mutations: S3T, V4I , S9R, A15T,K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,RS103A, V1041,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A,G160D, Y167A, R170S, A194P, G195E, V199M, V205I , L217D, N218D, M222S,A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN′ numbering).

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase®Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®,Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra,Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under thetradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®,Purafect MAO, Purafect Ox®, Purafect Ox®, Puramax®, Properase®, FN2®,FN3®, FN4®, Excellase®, Eraser®, Ultimase®, Opticlean® and Optimase®(Danisco/DuPont), Axapem™ (Gist-Brocases N.V.), BLAP (sequence shown inFIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof (Henkel AG) andKAP (Bacillus alkalophilus subtilisin) from Kao.

Lipases and Cutinases

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutant enzymes areincluded. Examples include lipase from Thermomyces, e.g. from T.lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP 305216, cutinase from Humicola, e.g. H. insolens (WO96/13580), lipase from strains of Pseudomonas (some of these now renamedto Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP 331376), P. sp. strain SD705 (WO 95/06720 & WO96/27002), P. wisconsinensis (WO 96/12012), GDSL-type Streptomyceslipases (WO 10/065455), cutinase from Magnaporthe grisea (WO 10/107560),cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipasefrom Thermobifida fusca (WO 11/084412), Geobacillus stearothermophiluslipase (WO 11/084417), lipase from Bacillus subtilis (WO 11/084599), andlipase from Streptomyces griseus (WO 11/150157) and S. pristinaespiralis(WO 12/137147).

Other examples are lipase variants such as those described in EP 407225,WO 92/05249, WO 94/01541, WO 94/25578, WO 95/14783, WO 95/30744, WO95/35381, WO 95/22615, WO 96/00292, WO 97/04079, WO 97/07202, WO00/34450, WO 00/60063, WO 01/92502, WO 07/87508 and WO 09/109500.

Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) andLipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g. acyltransferases with homologyto Candida antarctica lipase A (WO 10/111143), acyltransferase fromMycobacterium smegmatis (WO 05/56782), perhydrolases from the CE 7family (WO 09/67279), and variants of the M. smegmatis perhydrolase inparticular the S54V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd (WO 10/100028).

Amylases

Suitable amylases which can be used together with subtilase variants ofthe invention may be an alpha-amylase or a glucoamylase and may be ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of Bacilluslicheniformis, described in more detail in GB 1,296,839.

Suitable amylases include amylases having SEQ ID NO: 3 in WO 95/10603 orvariants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferredvariants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQID NO: 4 of WO 99/019467, such as variants with substitutions in one ormore of the following positions: 15, 23, 105, 106, 124, 128, 133, 154,156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243,264, 304, 305, 391, 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 in WO02/010355 or variants thereof having 90% sequence identity thereto.Preferred variants are those having a deletion in positions 181 and 182and a substitution in position 193.

Other amylases which are suitable are hybrid alpha-amylase comprisingresidues 1-33 of the alpha-amylase derived from B. amyloliquefaciensshown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B.licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 orvariants having 90% sequence identity thereof. Preferred variants ofthis hybrid alpha-amylase are those having a substitution, a deletion oran insertion in one of more of the following positions: G48, T49, G107,H156, A181, N190, M197, 1201, A209 and Q264. Most preferred variants ofthe hybrid alpha-amylase comprising residues 1-33 of the alpha-amylasederived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO2006/066594 and residues 36-483 of SEQ ID NO: 4 of WO2006/066594 arethose having the substitutions:

M197T;

H156Y+A181T+N190F+A209V+Q264S; or

G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

Further amylases which are suitable are amylases having SEQ ID NO: 6 inWO 99/019467 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Preferred variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, 1206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsR181 and G182, or positions H183 and G184.

Additional amylases which can be used are those having SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variantsthereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, adeletion or an insertion in one or more of the following positions: 140,181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476. Morepreferred variants are those having a deletion in positions 181 and 182or positions 183 and 184. Most preferred amylase variants of SEQ ID NO:1, SEQ ID NO: 2 or SEQ ID NO: 7 are those having a deletion in positions183 and 184 and a substitution in one or more of positions 140, 195,206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 of WO08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90% sequenceidentity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ IDNO: 10 in WO 01/66712 are those having a substitution, a deletion or aninsertion in one of more of the following positions: 176, 177, 178, 179,190, 201, 207, 211 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 of WO09/061380 or variants having 90% sequence identity to SEQ ID NO: 2thereof. Preferred variants of SEQ ID NO: 2 are those having atruncation of the C-terminus and/or a substitution, a deletion or aninsertion in one of more of the following positions: Q87, Q98, S125,N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243,N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferredvariants of SEQ ID NO: 2 are those having the substitution in one ofmore of the following positions: Q87E,R, Q98R, S125A, N128C, T131I ,T1651, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R,R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180and/or S181 or of T182 and/or G183. Most preferred amylase variants ofSEQ ID NO: 2 are those having the substitutions:

N128C+K178L+T182G+Y305R+G475K;

N1280+K178L+T182G+F202Y+Y305R+D319T+G475K;

S125A+N128C+K178L+T182G+Y305R+G475K; or

S125A+N128C+T131I+T1651+K178L+T182G+Y305R+G475K wherein the variants areC-terminally truncated and optionally further comprises a substitutionat position 243 and/or a deletion at position 180 and/or position 181.

Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90% sequence identity to SEQ IDNO: 12. Preferred amylase variants are those having a substitution, adeletion or an insertion in one of more of the following positions ofSEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181, G182, D183, G184,G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320,H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484.Particular preferred amylases include variants having a deletion of D183and G184 and having the substitutions R118K, N195F, R320K and R458K, anda variant additionally having substitutions in one or more positionselected from the group: M9, G149, G182, G186, M202, T257, Y295, N299,M323, E345 and A339, most preferred a variant that additionally hassubstitutions in all these positions.

Other examples are amylase variants such as those described in WO2011/098531, WO 2013/001078 and WO 2013/001087.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™,Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ (fromNovozymes A/S), and

Rapidase™, Purastar™/Effectenz™, Powerase and Preferenz S100 (fromGenencor International Inc./DuPont).

Peroxidases/Oxidases

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additive,i.e., a separate additive or a combined additive, can be formulated, forexample, as a granulate, liquid, slurry, etc. Preferred detergentadditive formulations are granulates, in particular non-dustinggranulates, liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

Adjunct Materials

Any detergent components known in the art for use in laundry detergentsmay also be utilized. Other optional detergent components includeanti-corrosion agents, anti-shrink agents, anti-soil redepositionagents, anti-wrinkling agents, bactericides, binders, corrosioninhibitors, disintegrants/disintegration agents, dyes, enzymestabilizers (including boric acid, borates, CMC, and/or polyols such aspropylene glycol), fabric conditioners including clays,fillers/processing aids, fluorescent whitening agents/opticalbrighteners, foam boosters, foam (suds) regulators, perfumes,soil-suspending agents, softeners, suds suppressors, tarnish inhibitors,and wicking agents, either alone or in combination. Any ingredient knownin the art for use in laundry detergents may be utilized. The choice ofsuch ingredients is well within the skill of the artisan.

Dispersants: The detergent compositions of the present invention canalso contain dispersants. In particular powdered detergents may comprisedispersants. Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms. Suitable dispersants are for exampledescribed in Powdered Detergents, Surfactant science series volume 71,Marcel Dekker, Inc.

Dye Transfer Inhibiting Agents: The detergent compositions of thepresent invention may also include one or more dye transfer inhibitingagents. Suitable polymeric dye transfer inhibiting agents include, butare not limited to, polyvinylpyrrolidone polymers, polyamine N-oxidepolymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole,polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. Whenpresent in a subject composition, the dye transfer inhibiting agents maybe present at levels from about 0.0001% to about 10%, from about 0.01%to about 5% or even from about 0.1% to about 3% by weight of thecomposition.

Fluorescent whitening agent: The detergent compositions of the presentinvention will preferably also contain additional components that maytint articles being cleaned, such as fluorescent whitening agent oroptical brighteners. Where present the brightener is preferably at alevel of about 0,01% to about 0,5%. Any fluorescent whitening agentsuitable for use in a laundry detergent composition may be used in thecomposition of the present invention. The most commonly used fluorescentwhitening agents are those belonging to the classes ofdiaminostilbene-sulphonic acid derivatives, diarylpyrazoline derivativesand bisphenyl-distyryl derivatives. Examples of thediaminostilbene-sulphonic acid derivative type of fluorescent whiteningagents include the sodium salts of:4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulphonate; 4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2.2′-disulphonate;4,4′-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2′-disulphonate;4,4′-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate and2-(stilbyl-4″-naptho-1.,2′:4,5)-1,2,3-trizole-2″-sulphonate. Preferredfluorescent whitening agents are Tinopal DMS and Tinopal CBS availablefrom Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium saltof 4,4′-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbenedisulphonate. Tinopal CBS is the disodium salt of2,2′-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescentwhitening agents is the commercially available Parawhite KX, supplied byParamount Minerals and Chemicals, Mumbai, India. Other fluorescerssuitable for use in the invention include the 1-3-diaryl pyrazolines andthe 7-alkylaminocoumarins. Suitable fluorescent brightener levelsinclude lower levels of from about 0.01, from 0.05, from about 0.1 oreven from about 0.2 wt % to upper levels of 0.5 or even 0.75 wt %.

Soil release polymers: The detergent compositions of the presentinvention may also include one or more soil release polymers which aidthe removal of soils from fabrics such as cotton and polyester basedfabrics, in particular the removal of hydrophobic soils from polyesterbased fabrics. The soil release polymers may for example be nonionic oranionic terephthalte based polymers, polyvinyl caprolactam and relatedcopolymers, vinyl graft copolymers, polyester polyamides see for exampleChapter 7 in Powdered Detergents, Surfactant science series volume 71,Marcel Dekker, Inc. Another type of soil release polymers areamphiphilic alkoxylated grease cleaning polymers comprising a corestructure and a plurality of alkoxylate groups attached to that corestructure. The core structure may comprise a polyalkylenimine structureor a polyalkanolamine structure as described in detail in WO 2009/087523(hereby incorporated by reference). Furthermore random graft co-polymersare suitable soil release polymers Suitable graft co-polymers aredescribed in more detail in WO 2007/138054, WO 2006/108856 and WO2006/113314 (hereby incorporated by reference). Other soil releasepolymers are substituted polysaccharide structures especiallysubstituted cellulosic structures such as modified cellulosederiviatives such as those described in EP 1867808 or WO 2003/040279(both are hereby incorporated by reference). Suitable cellulosicpolymers include cellulose, cellulose ethers, cellulose esters,cellulose amides and mixtures thereof. Suitable cellulosic polymersinclude anionically modified cellulose, nonionically modified cellulose,cationically modified cellulose, zwitterionically modified cellulose,and mixtures thereof. Suitable cellulosic polymers include methylcellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethylcellulose, hydroxyl propyl methyl cellulose, ester carboxy methylcellulose, and mixtures thereof.

Anti-redeposition agents: The detergent compositions of the presentinvention may also include one or more anti-redeposition agents such ascarboxymethylcellulose (CMC), polyvinyl alcohol (PVA),polyvinylpyrrolidone (PVP), polyoxyethylene and/or polyethyleneglycol(PEG), homopolymers of acrylic acid, copolymers of acrylic acid andmaleic acid, and ethoxylated polyethyleneimines. The cellulose basedpolymers described under soil release polymers above may also functionas anti-redeposition agents.

Other suitable adjunct materials include, but are not limited to,anti-shrink agents, anti-wrinkling agents, bactericides, binders,carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foamregulators, hydrotropes, perfumes, pigments, sod suppressors, solvents,and structurants for liquid detergents and/or structure elasticizingagents.

Formulation of Detergent Products

The detergent composition may be in any convenient form, e.g., a bar, ahomogenous tablet, a tablet having two or more layers, a pouch havingone or more compartments, a regular or compact powder, a granule, apaste, a gel, or a regular, compact or concentrated liquid. There are anumber of detergent formulation forms such as layers (same or differentphases), pouches, as well as forms for machine dosing unit.

Pouches may be configured as single or multicompartments. It may be ofany form, shape and material which is suitable for hold the composition,e.g. without allowing the release of the composition from the pouchprior to water contact. The pouch is made from water soluble film whichencloses an inner volume. Said inner volume may be divided intocompartments of the pouch. Preferred films are polymeric materialspreferably polymers which are formed into a film or sheet. Preferredpolymers, copolymers or derivates thereof are selected polyacrylates,and water soluble acrylate copolymers, methyl cellulose, carboxy methylcellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose,hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, mostpreferably polyvinyl alcohol copolymers and, hydroxyprpyl methylcellulose (HPMC). Preferably the level of polymer in the film forexample PVA is at least about 60%. Preferred average molecular weightwill typically be about 20,000 to about 150,000. Films can also be ofblend compositions comprising hydrolytically degradable and watersoluble polymer blends such as polyactide and polyvinyl alcohol (knownunder the Trade reference M8630 as sold by Chris Craft In. Prod. OfGary, Ind., US) plus plasticisers like glycerol, ethylene glycerol,Propylene glycol, sorbitol and mixtures thereof. The pouches maycomprise a solid laundry detergent composition or part components and/ora liquid cleaning composition or part components separated by the watersoluble film. The compartment for liquid components can be different incomposition than compartments containing solids. Ref: (US 2009/0011970A1).

Detergent ingredients can be separated physically from each other bycompartments in water dissolvable pouches or in different layers oftablets. Thereby negative storage interaction between components can beavoided. Different dissolution profiles of each of the compartments canalso give rise to delayed dissolution of selected components in the washsolution.

A liquid or gel detergent, which is not unit dosed, may be aqueous,typically containing at least 20% by weight and up to 95% water, such asup to about 70% water, up to about 65% water, up to about 55% water, upto about 45% water, up to about 35% water. Other types of liquids,including without limitation, alkanols, amines, diols, ethers andpolyols may be included in an aqueous liquid or gel. An aqueous liquidor gel detergent may contain from 0-30% organic solvent. A liquid or geldetergent may be non-aqueous.

Laundry Soap Bars

The subtilase variants of the invention may be added to laundry soapbars and used for hand washing laundry, fabrics and/or textiles. Theterm laundry soap bar includes laundry bars, soap bars, combo bars,syndet bars and detergent bars. The types of bar usually differ in thetype of surfactant they contain, and the term laundry soap bar includesthose containing soaps from fatty acids and/or synthetic soaps. Thelaundry soap bar has a physical form which is solid and not a liquid,gel or a powder at room temperature. The term solid is defined as aphysical form which does not significantly change over time, i.e. if asolid object (e.g. laundry soap bar) is placed inside a container, thesolid object does not change to fill the container it is placed in. Thebar is a solid typically in bar form but can be in other solid shapessuch as round or oval.

The laundry soap bar may contain one or more additional enzymes,protease inhibitors such as peptide aldehydes (or hydrosulfite adduct orhemiacetal adduct), boric acid, borate, borax and/or phenylboronic acidderivatives such as 4-formylphenylboronic acid, one or more soaps orsynthetic surfactants, polyols such as glycerine, pH controllingcompounds such as fatty acids, citric acid, acetic acid and/or formicacid, and/or a salt of a monovalent cation and an organic anion, whereinthe monovalent cation may be for example Na⁺, K⁺ or NH₄ ⁺ and theorganic anion may be for example formate, acetate, citrate or lactatesuch that the salt of a monovalent cation and an organic anion may be,for example, sodium formate.

The laundry soap bar may also contain complexing agents like EDTA andHEDP, perfumes and/or different type of fillers, surfactants e.g.anionic synthetic surfactants, builders, polymeric soil release agents,detergent chelators, stabilizing agents, fillers, dyes, colorants, dyetransfer inhibitors, alkoxylated polycarbonates, suds suppressers,structurants, binders, leaching agents, bleaching activators, clay soilremoval agents, anti-redeposition agents, polymeric dispersing agents,brighteners, fabric softeners, perfumes and/or other compounds known inthe art.

The laundry soap bar may be processed in conventional laundry soap barmaking equipment such as but not limited to: mixers, plodders, e.g. atwo stage vacuum plodder, extruders, cutters, logo-stampers, coolingtunnels and wrappers. The invention is not limited to preparing thelaundry soap bars by any single method. The premix may be added to thesoap at different stages of the process. For example, the premixcontaining a soap, an enzyme, optionally one or more additional enzymes,a protease inhibitor, and a salt of a monovalent cation and an organicanion may be prepared and the mixture is then plodded. The enzyme andoptional additional enzymes may be added at the same time as theprotease inhibitor for example in liquid form. Besides the mixing stepand the plodding step, the process may further comprise the steps ofmilling, extruding, cutting, stamping, cooling and/or wrapping.

Granular Detergent Formulations

A granular detergent may be formulated as described in WO09/092699,EP1705241, EP1382668, WO07/001262, U.S. Pat. No. 6,472,364, WO04/074419or WO09/102854. Other useful detergent formulations are described inWO09/124162, WO09/124163, WO09/117340, WO09/117341, WO09/117342,WO09/072069, WO09/063355, WO09/132870, WO09/121757, WO09/112296,WO09/112298, WO09/103822, WO09/087033, WO09/050026, WO09/047125,WO09/047126, WO09/047127, WO09/047128, WO09/021784, WO09/010375,WO09/000605, WO09/122125, WO09/095645, WO09/040544, WO09/040545,WO09/024780, WO09/004295, WO09/004294, WO09/121725, WO09/115391,WO09/115392, WO09/074398, WO09/074403, WO09/068501, WO09/065770,WO09/021813, WO09/030632, and WO09/015951.

WO2011025615, WO2011016958, WO2011005803, WO2011005623, WO2011005730,WO2011005844, WO2011005904, WO2011005630, WO2011005830, WO2011005912,WO2011005905, WO2011005910, WO2011005813, WO2010135238, WO2010120863,WO2010108002, WO2010111365, WO2010108000, WO2010107635, WO2010090915,WO2010033976, WO2010033746, WO2010033747, WO2010033897, WO2010033979,WO2010030540, WO2010030541, WO2010030539, WO2010024467, WO2010024469,WO2010024470, WO2010025161, WO2010014395, WO2010044905,

WO2010145887, WO2010142503, WO2010122051, WO2010102861, WO2010099997,WO2010084039, WO2010076292, WO2010069742, WO2010069718, WO2010069957,WO2010057784, WO2010054986, WO2010018043, WO2010003783, WO2010003792,

WO2011023716, WO2010142539, WO2010118959, WO2010115813, WO2010105942,WO2010105961, WO2010105962, WO2010094356, WO2010084203, WO2010078979,WO2010072456, WO2010069905, WO2010076165, WO2010072603, WO2010066486,WO2010066631, WO2010066632, WO2010063689, WO2010060821, WO2010049187,WO2010031607, WO2010000636.

Uses

The subtilase variants according to the invention or compositionsthereof may be used in laundering of textile and fabrics, such as household laundry washing and industrial laundry washing.

The subtilase variants according to the invention or compositionsthereof may also be used in methods for cleaning hard surfaces such asfloors, tables, walls, roofs etc. as well as surfaces of hard objectssuch as cars (car wash) and dishes (dish wash).

A detergent composition may be formulated, for example, as a hand ormachine laundry detergent composition including a laundry additivecomposition suitable for pre-treatment of stained fabrics and a rinseadded fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

The polypeptides of the present invention may be added to a detergentadditive.

The cleaning process or the textile care process may for example be alaundry process, a dishwashing process or cleaning of hard surfaces suchas bathroom tiles, floors, table tops, drains, sinks and washbasins.Laundry processes may for example be household laundering, but it mayalso be industrial laundering. Processes for laundering of fabricsand/or garments where the process comprises treating fabrics with awashing solution containing a detergent composition may be added atleast one subtilase variant of the invention. The cleaning process or atextile care process may for example be carried out in a machine washingprocess or in a manual washing process. The washing solution may forexample be an aqueous washing solution containing a detergentcomposition.

The last few years there has been an increasing interest in replacingcomponents in detergents, which is derived from petrochemicals withrenewable biological components such as enzymes and polypeptides withoutcompromising the wash performance. When the components of detergentcompositions change new enzyme activities or new enzymes havingalternative and/or improved properties compared to the common useddetergent enzymes such as proteases, lipases and amylases is needed toachieve a similar or improved wash performance when compared to thetraditional detergent compositions.

The invention further concerns the use of subtilase variants of theinvention in a proteinaceous stain removing processes. The proteinaceousstains may be stains such as food stains, e.g., baby food, sebum, cocoa,egg, blood, milk, ink, grass, or a combination hereof.

Typical detergent compositions include various components in addition tothe enzymes, these components have different effects, some componentslike the surfactants lower the surface tension in the detergent, whichallows the stain being cleaned to be lifted and dispersed and thenwashed away, other components like bleach systems remove discolor oftenby oxidation and many bleaches also have strong bactericidal properties,and are used for disinfecting and sterilizing. Yet other components likebuilder and chelator softens, e.g., the wash water by removing the metalions form the liquid.

Composition comprising a subtilase variant of the invention may compriseone or more detergent components, such as surfactants, hydrotropes,builders, co-builders, chelators or chelating agents, bleaching systemor bleach components, polymers, fabric hueing agents, fabricconditioners, foam boosters, suds suppressors, dispersants, dye transferinhibitors, fluorescent whitening agents, perfume, optical brighteners,bactericides, fungicides, soil suspending agents, soil release polymers,anti-redeposition agents, enzyme inhibitors or stabilizers, enzymeactivators, antioxidants, and solubilizers.

A composition may comprising a subtilase variant of the invention andone or more additional enzymes selected from the group comprising ofproteases, amylases, lipases, cutinases, cellulases, endoglucanases,xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidaes,haloperoxygenases, catalases and mannanases, or any mixture thereof.

A composition may comprise a subtilase variant of the invention, one ormore additional enzymes selected from the group comprising of proteases,amylases, lipases, cutinases, cellulases, endoglucanases,xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidaes,haloperoxygenases, catalases and mannanases, or any mixture thereof andone or more detergent components, such as surfactants, hydrotropes,builders, co-builders, chelators or chelating agents, bleaching systemor bleach components, polymers, fabric hueing agents, fabricconditioners, foam boosters, suds suppressors, dispersants, dye transferinhibitors, fluorescent whitening agents, perfume, optical brighteners,bactericides, fungicides, soil suspending agents, soil release polymers,anti-redeposition agents, enzyme inhibitors or stabilizers, enzymeactivators, antioxidants, and solubilizers.

Washing Method

A method of cleaning may comprise the steps of: contacting an objectwith a detergent composition comprising a subtilase variant of theinvention under conditions suitable for cleaning said object.

A method for removing stains from fabric or dishware may comprisecontacting said fabric or dishware with a composition comprising asubtilase variant of the invention under conditions suitable forcleaning said object.

Compositions and methods of treating fabrics (e.g., to desize a textile)may use one or more of the subtilase variant of the invention. Thevariant may be used in any fabric-treating method which is well-known inthe art (see, e.g., U.S. Pat. No. 6,077,316). For example, in oneaspect, the feel and appearance of a fabric is improved by a methodcomprising contacting the fabric with a protease in a solution. In oneaspect, the fabric is treated with the solution under pressure.

Detergent compositions are suited for use in laundry and hard surfaceapplications, including dish wash. Such methods may comprise the stepsof contacting the fabric/dishware to be cleaned with a solutioncomprising a detergent composition. The fabric may comprise any fabriccapable of being laundered in normal consumer use conditions. Thedishware may comprise any dishware such as crockery, cutlery, ceramics,plastics such as melamine, metals, china, glass and acrylics. Thesolution preferably has a pH from about 5.5 to about 11.5. Thecompositions may be employed at concentrations from about 100 ppm,preferably 500 ppm to about 15,000 ppm in solution. The watertemperatures typically range from about 5° C. to about 95° C., includingabout 10° C., about 15° C., about 20° C., about 25° C., about 30° C.,about 35° C., about 40° C., about 45° C., about 50° C., about 55° C.,about 60° C., about 65° C., about 70° C., about 75° C., about 80° C.,about 85° C. and about 90° C. The water to fabric ratio is typicallyfrom about 1:1 to about 30:1.

The enzyme(s) of the detergent composition may be stabilized usingconventional stabilizing agents and protease inhibitors, e.g. a polyolsuch as propylene glycol or glycerol, a sugar or sugar alcohol,different salts such as NaCl; KCl; lactic acid, formic acid, boric acid,or a boric acid derivative, e.g., an aromatic borate ester, or a phenylboronic acid derivative such as 4-formylphenyl boronic acid, or apeptide aldehyde such as di-, tri- or tetrapeptide aldehydes or aldehydeanalogues (either of the form B1-B0-R wherein, R is H, CH3, CX3, CHX2,or CH2X (X=halogen), B0 is a single amino acid residue (preferably withan optionally substituted aliphatic or aromatic side chain); and B1consists of one or more amino acid residues (preferably one, two orthree), optionally comprising an N-terminal protection group, or asdescribed in WO 09118375, WO 98/13459) or a protease inhibitor of theprotein type such as RASI, BASI, WASI (bifunctionalalpha-amylase/subtilisin inhibitors of rice, barley and wheat) or C12 orSSI. The composition may be formulated as described in e.g. WO 92/19709,WO 92/19708 and U.S. Pat. No. 6,472,364. In some embodiments, theenzymes employed herein are stabilized by the presence of water-solublesources of zinc (II), calcium (II) and/or magnesium (II) ions in thefinished compositions that provide such ions to the enzymes, as well asother metal ions (e.g., barium (II), scandium (II), iron (II), manganese(II), aluminum (III), Tin (II), cobalt (II), copper (II), Nickel (II),and oxovanadium (IV)).

The detergent compositions are typically formulated such that, duringuse in aqueous cleaning operations, the wash water has a pH of fromabout 5.0 to about 11.5, or in alternative embodiments, even from about6.0 to about 10.5. In some preferred embodiments, granular or liquidlaundry products are formulated to have a pH from about 6 to about 8.Techniques for controlling pH at recommended usage levels include theuse of buffers, alkalis, acids, etc., and are well known to thoseskilled in the art.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Materials and Methods

Automatic Mechanical Stress Assay (AMSA) for laundry

In order to assess the wash performance in laundry, washing experimentsare performed, using the Automatic Mechanical Stress Assay (AMSA). WithAMSA, the wash performance of a large quantity of small volumeenzyme-detergent solutions can be examined. The AMSA plate has a numberof slots for test solutions and a lid firmly squeezing the laundrysample, the textile to be washed against all the slot openings. Duringthe washing time, the plate, test solutions, textile and lid arevigorously shaken to bring the test solution in contact with the textileand apply mechanical stress in a regular, periodic oscillating manner.For further description see WO02/42740 especially the paragraph “Specialmethod embodiments” at page 23-24.

The wash performance is measured as the brightness of the colour of thetextile washed. Brightness can also be expressed as the intensity of thelight reflected from the sample when illuminated with white light. Whenthe sample is stained the intensity of the reflected light is lower,than that of a clean sample. Therefore, the intensity of the reflectedlight can be used to measure wash performance.

Colour measurements are made with a professional flatbed scanner (KodakiQsmart, Kodak, Midtager 29, DK-2605 Brøndby, Denmark), which is used tocapture an image of the washed textile.

To extract a value for the light intensity from the scanned images,24-bit pixel values from the image are converted into values for red,green and blue (RGB). The intensity value (Int) is calculated by addingthe RGB values together as vectors and then taking the length of theresulting vector:

Int=√{square root over (r ² +g ² +b ²)}

Table 1: Composition of model detergents and test materials

Model detergent and test materials were as follows:

Laundry liquid 0.3 to 0.5% xanthan gum, model detergent 0.2 to 0.4%antifoaming agent, 6 to 7% glycerol, 0.3 to 0.5% ethanol, 4 to 7% FAEOS(fatty alcohol ether sulfate), 24 to 28% nonionic surfactants, 1% boricacid, 1 to 2% sodium citrate (dihydrate), 2 to 4% soda, 14 to 16%coconut fatty acid, 0.5% HEDP (1-hydroxyethane-(1.1- diphosphonicacid)), 0 to 0.4% PVP (polyvinylpyrrolidone), 0 to 0.05% opticalbrighteners, 0 to 0.001% dye, remainder deionized water. Test materialPC-03 (Chocolate-milk/ink on cotton/polyester) C-05 (Blood/milk/ink oncotton)

Tergo-O-Tometer (TOM)

The Tergo-O-Tometer (TOM) is a medium scale model wash system that canbe applied to test 12 different wash conditions simultaneously. A TOM isbasically a large temperature controlled water bath with up to 12 openmetal beakers submerged into it. Each beaker constitutes one small toploader style washing machine and during an experiment, each of them willcontain a solution of a specific detergent/enzyme system and the soiledand unsoiled fabrics its performance is tested on. Mechanical stress isachieved by a rotating stirring arm, which stirs the liquid (1L) withineach beaker. Because the TOM beakers have no lid, it is possible towithdraw samples during a TOM experiment and assay for informationon-line during wash. In a TOM experiment, factors such as the ballast tosoil ratio and the fabric to wash liquor ratio can be varied. Therefore,the TOM provides the link between small scale experiments, such as AMSAand mini-wash, and the more time consuming full scale experiments infull scale washing machines.

After washing and rinsing the swatches are spread out flat and allowedto air dry at room temperature overnight. All washes are evaluated theday after the wash. Light reflectance evaluations of the swatches aredone using a Macbeth Color Eye 7000 reflectance spectrophotometer withlarge aperture. The measurements are made without UV in the incidentlight and remission at 460 nm was extracted. Measurements are made onunwashed and washed swatches. The test swatch to be measured is placedon top of another swatch of same type and colour (twin swatch).

Remission values for individual swatches are calculated by subtractingthe remission value of the swatch washed without enzyme (blank) from theswatch washed together with enzyme.

Calculating the effect of protease variants effect is done by taking themeasurements from washed swatches with enzymes and subtracting themeasurements from washed without enzyme for each stain.

The performance of the new protease variants is compared to theperformance of Reference (REF)=SEQ ID NO: 3 by calculating the relativeperformance (RP):

RP=(R _(PROTEASEVARIANT) −R _(BLANK))/(R _(REF) −R _(BLANK))

General Molecular Biology Methods:

Unless otherwise mentioned the DNA manipulations and transformationswere performed using standard methods of molecular biology (Sambrook etal. (1989); Ausubel et al. (1995); Harwood and Cutting (1990).

Protease Activity Assay:

1) Suc-AAPF-pNA activity assay:

The proteolytic activity can be determined by a method employing theSuc-AAPF-PNA substrate. Suc-AAPF-PNA is an abbreviation forN-Succinyl-Alanine-Alanine-Proline-Phenylalanine-p-Nitroanilide, and itis a blocked peptide which can be cleaved by endo-proteases. Followingcleavage a free PNA molecule is liberated and it has a yellow colour andthus can be measured by visible spectrophotometry at wavelength 405 nm.The Suc-AAPF-PNA substrate is manufactured by Bachem (cat. no. L1400,dissolved in DMSO).

The protease sample to be analyzed was diluted in residual activitybuffer (100 mM Tris pH8.6). The assay was performed by transferring 60μl of diluted enzyme samples to 96 well microtiter plate and adding 140μl substrate working solution (0.72 mg/ml in 100 mM Tris pH8.6). Thesolution was mixed at room temperature and absorption is measured every20 sec. over 5 minutes at OD 405 nm.

The slope (absorbance per minute) of the time dependent absorption-curveis directly proportional to the specific activity (activity per mgenzyme) of the protease in question under the given set of conditions.The protease sample should be diluted to a level where the slope islinear.

Accelerated Storage Stability Assay

Storage stability of protease variants in liquid detergent was evaluatedusing an accelerated assay with incubation at elevated temperatures forup to 24 hours.

All purified protease samples were diluted to concentrations of 0.2 and0.1 mg/ml based on absorbance at 280 nm and theoretical extinctioncoefficient using 0.01% Triton X-100. For each variant 2 wells with highprotease concentration and 2 wells with low concentration were included.As reference SEQ ID NO: 3 was included on each microtiter plate. 30 μlprotease sample was mixed with 270 μl detergent (CNS, EDTA pH9) in thewell of a microtiter plate (Nunc U96 PP 0.5 ml) using a magnetic bar (onZephyr pipetting station (Caliper LifeSciences) for 30 min). 20 μl ofthis mixture was then transferred to another microtiter plate (Nunc U96PP 0.5 ml with added magnetic bars) and mixed with 150 μl 100 mM Tris pH8.6 (at least 5 min on Zephyr). 30 μl of this dilution was transferredto a Nunc F 96-MTP, and after addition of 70 μl substrate solutioninitial activity of unstressed sample was determined by measuringabsorbance at 405 nm every 20 sec for 5 min (on a SpectraMax Plus).After sealing, the detergent plate was incubated at appropriatetemperature (47° C. for CNS, EDTA pH9) in an Eppendorf Thermomixer (noshaking). After 1-4 and 20-25 hours incubation, 20 μl samples werewithdrawn and residual activity of stressed sample was measured as withthe initial, unstressed activity.

Decrease in activity during incubation with detergent was assumed to beexponential. Half lifes (T½) were found from linear regression ofLog(Activity) versus incubation time (0, 1-4 and 20-25 hours), andhalf-life improvement factors (T½ IF) were calculated as half-life ofprotease variant relative to half-life of SEQ ID NO: 3 reference.

Detergent

CNS Alkylbenzenesulfonic acid 8% EDTA Alcohol ethoxylate 4% pH9 Sodiumlauryl ether sulfate 4% Triethanolamine; 2,2′,2″-nitrilotri(ethan-1-ol)0.5% Trisodium citrate dihydrate 0.5% Sodium hydroxide 1% EDTA 0.001% pHadjusted to 9 Up to 100% demineralised water

Example 1: Preparation and Expression of Variants

Introduction of an expression cassette into Bacillus subtilis was doneby transformation of a suitable expression cassette utilizing thenatural competence of the organism.

DNA manipulations such as The introduction of mutations and constructionof an expression cassette into Bacillus subtilis were done by PCR (e.g.Sambrook et al.; Molecular Cloning; Cold Spring Harbor Laboratory Press)and all DNA manipulations and can be repeated by everybody skilled inthe art. Recombinant B. subtilis constructs encoding subtilase variantswere used to inoculate shakeflasks containing a rich media (e.g. PS-1:100 g/L Sucrose (Danisco cat.no. 109-0429), 40 g/L crust soy (soy beanflour), 10 g/L Na2HPO4.12H2O (Merck cat.no. 6579), 0.1 ml/Lreplace-Dowfax63N10 (Dow). Cultivation typically takes 4 days at 30° C.shaking with 220 rpm.

Example 2: Fermentation of Variants

Fermentation may be performed by methods well known in the art or asfollows. A B. subtilis strain harboring the relevant expression plasmidwas streaked on a LB agar plate, and grown overnight at 37° C. Thecolonies were transferred to 100 ml PS-1 media in a 500 ml shakingflask. Cells and other undissolved material were removed from thefermentation broth by centrifugation at 4500 rpm for 20-25 minutes.Afterwards the supernatant was filtered to obtain a clear solution.

Example 3: Purification of Variants

The culture broth was centrifuged (26000×g, 20 min) and the supernatantwas carefully decanted from the precipitate. The supernatant wasfiltered through a Nalgene 0.2 μm filtration unit in order to remove therest of the Bacillus host cells. pH in the 0.2 μm filtrate was adjustedto pH 8 with 3M Tris base and the pH adjusted filtrate was applied to aMEP Hypercel column (from Pall corporation) equilibrated in 20 mMTris/HCl, 1 mM CaCl₂, pH 8.0. After washing the column with theequilibration buffer, the column was step-eluted with 20 mMCH₃COOH/NaOH, 1 mM CaCl₂, pH 4.5. Fractions from the column wereanalysed for protease activity (using the Suc-AAPF-pNA assay at pH 9)and peak-fractions were pooled. The pH of the pool from the MEP Hypercelcolumn was adjusted to pH 6 with 20% (v/v) CH₃COOH or 3M Tris base andthe pH adjusted pool was diluted with deionized water to the sameconductivity as 20 mM MES/NaOH, 2 mM CaCl₂, pH 6.0. The diluted pool wasapplied to a SP-sepharose FF column (from GE Healthcare) equilibrated in20 mM MES/NaOH, 2 mM CaCl₂, pH 6.0. After washing the column with theequilibration buffer, the protease was eluted with a linear NaClgradient (0-->0.5M) in the same buffer over five column volumes.Fractions from the column were analysed for protease activity (using theSuc-AAPF-pNA assay at pH 9) and active fractions were analysed bySDS-PAGE. The fractions, where only one band was seen on the coomassiestained SDS-PAGE gel, were pooled as the purified preparation and wasused for further experiments.

Example 4: Stability of Variants of the Invention

Variants of the invention were generated and purified as described inexamples 1-3 and tested for stability in liquid detergent at atemperature of 47° C. using the stability test disclosed above and thehalf-life calculated. The reference polypeptide was the subtilase havingSEQ ID NO: 3.

TABLE 2 Stability of variants of SEQ ID NO: 3. First column indicate thesubstitution in SEQ ID NO: 3. Second column indicate the half-life foundin the experiment, and in third column the data are indicated relativeto SEQ ID NO: 3, Last column indicate the standard deviation:Substitutions T½ (h) IF (47° C., StDev (Relative to (47° C., 24 h, 24 h,90% (47° C., 24 h, SEQ ID NO : 3) 90% detergent) detergent) 90%detergent) None 23 1 0.06 (=SEQ ID NO: 3) H120V 30 1.41 0.11 N261T 311.45 0.07 S163A 55 2.1 0.2 M222Q 51 2 0.1 R45E 53 2.1 0.2 N185Q 28 1.40.2 N117E 34 1.4 0.1 S87E 58 2.1 0.2 L124M 28 1.4 0.2 P129D 69 2.6 0.5R45Q 27 1.3 0.1 L262N 50 2.4 0.5 L262Q 47 2.2 0.3 V199M 27 1.2 0.1 S3T30 1.3 0.1 H120K 31 1.4 0.1 H120N 22 1 0.1 N238H 20 0.9 0.1 Q59M 21 0.90.1 A98D 21 0.9 0.1 G61R 20 0.9 0.1 G97S 24 0.8 0.1 A98E 24 0.8 0.1 A98R26 0.9 0.1 S163G 38 1.3 0.2

All the variants had on par stability, improved stability orsignificantly improved stability in liquid detergent under theseconditions.

Example 5 Residual Activity

Variants of the invention were tested for stability in liquid detergentat a temperature 45° C. using the stability test disclosed above and thehalf-life and residual activity after 19 hours calculated. The parentpolypeptide was the subtilase having SEQ ID NO: 3.

TABLE 3 Stability of variants of SEQ ID NO: 3. First column indicate thesubstitution compared to SEQ ID NO: 3. Second column indicate thecalculated residual activity after 19 hours and last column indicate theresidual activity with standard deviation: Substitutions % RA (Relativeto (45° C., 19 h, % RA ± Stdev SEQ ID NO: 3) 90% detergent) (45° C., 19h) None 75 75 ± 9 (=SEQ ID NO: 3) V199M 85 85 ± 3 S3T 90 90 ± 1 S106A 9999 ± 3 S106W 87 87 ± 7 T143W 81 81 ± 4 H120D 90 90 ± 3 H120K 97 97 ± 5H120N 84 84 ± 6 N238H 81 81 ± 3 P55N 86 86 ± 3 T58W 89 89 ± 5 T58Y 83 83± 4 Q59D 83 83 ± 4 Q59M 89 89 ± 6 Q59N 88 88 ± 3 Q59T 82 82 ± 4 G61D 8585 ± 3 A98D 81 81 ± 3 A172S 82 82 ± 7 V244T 86  86 ± 11 Y171L 86 86 ± 2G61R 82 82 ± 2 L262D 89 89 ± 2 S161T 91 91 ± 6 G97S 92 92 ± 3 A98E 90 90± 1 A98R 97 97 ± 2 E136Q 88 88 ± 3 S163G 104 104 ± 4  T58L 105 105 ± 2 

The data shows that these variants had increased stability under thetested conditions and also increased residual activity after 19 hourscompared with the parent subtilase having SEQ ID NO: 3.

Example 6

The wash performance of variants in detergents was determined by usingthe following standardized stains:

-   A: egg-yolk on cotton: product no. 10EG obtainable from W-Testgewebe    GmbH, Christenfeld 10, 41379 Bruggen, Germany-   B: blood on cotton: product no. CS01 obtainable from CFT (Center for    Testmaterials) B.V., Vlaardingen, Netherlands,-   C: egg on cotton: product no. C37 obtainable from CFT (Center for    Testmaterials) B.V., Vlaardingen, Netherlands,-   D: blood on cotton: product no. 111 obtainable from Eidgenössische    Material-und Prüfanstalt (EMPA) Testmaterialien AG [Federal    materials and testing agency, Testmaterials], St. Gallen,    Switzerland.-   The following stains E-R are all obtainable from CFT (Center for    Testmaterials) B.V., Vlaardingen, Netherlands:-   E: cocoa on cotton: product no. CH-09-   F: egg on cotton: product no. C38-   G: chocolate-milk on cotton: product no. CO3-   H: cocoa-oatmeal: product no. C-S-54-   I: chocolate-milk on polyester/cotton: product no. PC-3-009-   J: cocoa cooked with milk on cotton: product no. C-H019-   K: meal replacement shake on cotton: product no. C-H165-   L: chocolate pudding on cotton: product no. C-H118-   M: chocolate pudding on cotton: product no. C-H172-   N: meat pate vallette: product no. KC-H 171-   0: chocolate pudding on cotton: product no. KC-H 172-   P: chocolate ice cream aged on cotton: product no. CS-68-   Q: chocolate pudding on cotton: product no. CS-69-   R: egg yolk carbon black aged: product no. CS-38-   S: egg on cotton: product no. WFK 10N obtainable from W-Testgewebe    GmbH, Christenfeld 10, 41379 Brüggen, Germany-   T: cocoa on cotton: product no. EMPA 112 obtainable from    Eidgenössische Material-und Prüfanstalt (EMPA) Testmaterialien AG    [Federal materials and testing agency, Testmaterials], St. Gallen,    Switzerland.-   U: blood-milk/ink on cotton: product no.C05-   V peanut oil pigment/ink on cotton: product no.C10-   W: grass on cotton: product no. 164 obtainable from Eidgenössische    Material-und Prüfanstalt (EMPA) Testmaterialien AG [Federal    materials and testing agency, Testmaterials], St. Gallen,    Switzerland.-   X: cocoa cooked up with milk: product no. C-H010-   Y: blood/milk/ink, product no. EMPA 117 obtainable from    Eidgenössische Material-und Prüfanstalt (EMPA) Testmaterialien AG    [Federal materials and testing agency, Testmaterials], St. Gallen,    Switzerland.-   Z: chocolate milk and soot, product no. CFT C03 obtainable from CFT    (Center for Testmaterials) B.V., Vlaardingen, Netherlands:

A liquid washing agent with the following composition was used as baseformulation (all values in weight percent): 0 to 0.5% xanthan gum, 0.2to 0.4% antifoaming agent, 0.2 to 8% Triethanolamine, 1 to 7% glycerol,0.3 to 3% ethanol, 0 to 12% FAEOS (fatty alcohol ether sulfate), 1 to28% nonionic surfactants, 0.5-4% boric acid, 0.5 to 6% sodium citrate(dihydrate), 1 to 6% soda, 0 to 16% coconut fatty acid, 0.5 to 6% HEDP(1-hydroxyethane-(1,1-diphosphonic acid)), 0 to 0.4% PVP(polyvinylpyrrolidone), 0 to 0.05% optical brighteners, 0 to 0.001% dye,remainder deionized water.

Based on this base formulation, various detergents were prepared byadding respective proteases as indicated in tables 4. Reference is theprotease that has the amino acid sequence of SEQ ID NO. 3, the referenceprotease already showing a good wash performance, especially in liquiddetergents. The proteases were added in the same amounts based on totalprotein content (5 mg/I wash liquor).

The dosing ratio of the liquid washing agent was 4.7 grams per liter ofwashing liquor and the washing procedure was performed for 60 minutes ata temperature of 20° C. and 40° C., the water having a water hardnessbetween 15.5 and 16.5° (German degrees of hardness).

The whiteness, i.e. the brightening of the stains, was determinedphotometrically as an indication of wash performance. A Minolta CM508dspectrometer device was used, which was calibrated beforehand using awhite standard provided with the unit.

The results obtained are the difference values between the remissionunits obtained with the detergents and the remission units obtained withthe detergent containing the reference protease. A positive valuetherefore indicates an improved wash performance of the variants in thedetergent. It is evident from tables 4a (results at 40° C.) and 4b(results at 20° C.) that variants according to the invention showimproved wash performance.

TABLE 4a and b Wash performance at 40° C. of protease variants that havethe same amino acid sequence as SEQ ID NO: 3 except for thesubstitutions as per the table below on the stains as indicated;reference is the protease according to SEQ ID NO: 3. a) Protease variantA B C D E F N76D A228V N261D Diff 1.3 3.0 10.6 nd nd nd HSD 0.5 2.2 2.4nd nd nd H120D S163G N261D Diff 1.1 1.5 9.2 nd nd nd HSD 0.5 2.2 2.4 ndnd nd N76D A228V L262E Diff 1.3 1.3 9.6 nd nd nd HSD 0.5 2.2 2.4 nd ndnd S156D L262E Diff 1.4 3.9 5.8 7.5 3.0 5.6 HSD 0.7 1.9 2.2 3.7 1.3 3.7N76D Q137H S141H R145H A228V Diff 1.1 0.8 4.7 0.4 1.2 4.1 N261D HSD 0.71.9 2.2 3.7 1.2 3.7 H120D S163G N261D Q137H S141H Diff 1.0 1.8 5.0 2.21.0 4.4 R145H HSD 0.7 2.0 2.2 3.7 1.2 3.7 N76D S163G N238E Q137H S141HDiff 1.3 3.4 6.2 4.4 1.7 5.9 R145H HSD 0.7 1.9 2.2 3.7 1.2 3.7 N238EL262E Q137H S141H R145H Diff 1.4 3.3 5.6 3.6 1.3 5.3 HSD 0.7 1.9 2.2 3.71.2 3.7 S3T N76D S156D Y209W Q137H S141H Diff 1.7 3.3 5.6 4.6 1.4 4.0R145H HSD 0.7 1.9 2.2 3.7 1.2 3.7 b) Protease Variant U G V T W S163G0.9 1.5 1.8 nd 0.9 G61D 1.5 1.7 nd nd nd S156D 1.6 2.1 0.5 nd nd H120D0.8 1.6 2.4 nd nd G195E V199M 1.5 0.7 1.1 nd nd A228V N261D 0.5 1.3 1.6nd nd V244T 1.3 1.1 2.7 nd nd T58L nd 2.0 1.1 0.9 0.4 S3T V4I N261D 1.51.7 0.4 nd nd A194P G195E V199M V205I 0.8 nd nd 1.1 nd H120D A228V 2.11.9 nd nd nd H120D N261D nd nd 0.8 1.2 nd N76D A228V N261D nd 0.5 nd 1.8nd

TABLES 4 c-e Wash performance at 20° C. of protease variants that havethe same amino acid sequence as SEQ ID NO: 3 except for thesubstitutions as per the table below on the stains as indicated;reference is the protease according to SEQ ID NO: 3. c) Protease variantA C D G H I H120D N261D Diff 0.8 7.1 4.3 1.9 2.4 4.8 HSD 0.7 4.5 2.5 2.61.2 1.2 N76D A228V N261D Diff 1.0 9.9 4.9 4.5 2.6 5.4 HSD 0.7 4.5 2.52.6 1.2 1.1 d) Protease variant A C D J K L M N O P Q R S T S156D Diff0.9 8.0 6.1 5.1 2.3 3.1 4.2 2.6 6.1 1.3 1.9 3.2 2.6 0.6 L262E HSD 0.93.7 4.2 2.0 1.4 2.4 1.5 2.2 3.2 1.1 1.6 2.6 1.9 1.7 N76D Q137H Diff 0.25.2 3.5 4.3 1.9 1.6 3.7 0.5 5.6 1.5 0.7 2.3 1.3 0.1 S141H HSD 0.9 3.74.2 2.0 1.4 2.4 1.6 2.2 3.3 1.1 1.6 2.7 1.9 1.7 R145H A228V N261D H120DDiff 1.1 7.0 6.0 4.4 2.2 2.9 4.2 3.1 5.9 1.4 2.7 3.5 2.6 1.2 S163G HSD0.9 3.7 4.2 2.0 1.4 2.4 1.5 2.2 3.2 1.1 1.6 2.6 1.9 1.7 N261D Q137HS141H R145H N76D S163G Diff 1.5 6.3 5.5 4.7 2.1 3.0 3.5 3.5 7.0 1.5 3.43.6 3.2 2.6 N238E HSD 0.9 3.7 4.2 2.0 1.4 2.4 1.5 2.2 3.2 1.1 1.6 2.71.9 1.8 Q137H S141H R145H N238E Diff 1.4 5.7 5.9 3.9 1.9 1.5 3.4 1.4 5.80.9 1.2 3.6 2.2 0.7 L262E HSD 0.9 3.7 4.2 2.0 1.4 2.4 1.5 2.2 3.2 1.11.6 2.6 1.9 1.7 Q137H S141H R145H S3T N76D Diff 2.3 6.7 6.7 4.4 2.6 1.83.5 3.0 5.5 1.5 1.9 2.0 2.7 1.8 S156D HSD 0.9 3.7 4.2 2.0 1.4 2.4 1.52.2 3.2 1.1 1.7 2.6 1.9 1.7 Y209W Q137H S141H R145H e) Protease VariantU G V T W S163G 1.1 1.3 0.5 1.7 nd G61D 0.2 0.8 0.7 2.1 nd H120D nd 0.30.6 0.5 1.2 S156D 1.1 1.9 nd 2.1 0.8 H120D nd 0.8 nd 0.9 1.2 G195E V199M1.1 1.0 nd 1.2 1.0 A228V N261D 0.8 0.8 nd 1.0 0.6 V244T nd 1.4 0.5 nd1.5 T58L 1.1 1.2 nd nd nd S3T V4I N261D 1.1 1.0 nd nd nd A194P G195EV199M V205I 0.4 1.3 nd nd nd H120D A228V 1.8 1.4 nd 2.0 nd N76D N261D ndnd 1.3 2.0 nd A194P G195E V199M V205I A228V N261D 0.6 nd 1.4 1.6 nd S3TV4I A228V 1.7 nd 1.5 0.6 nd A194P G195E V205I A228V 0.8 nd 2.2 nd ndH120D N261D nd 1.9 nd nd nd N76D A228V N261D 0.7 4.5 0.8 1.3 nd f)Protease variant A C G L X Y Z N238E L262E Diff 1.2 4.6 0.6 3.9 5.5 1.23.1 HSD 0.9 2.4 2.0 2.6 2.5 3.3 2.5 S156D L262E Diff 1.5 5.7 0.8 3.2 5.82.6 4.5 HSD 0.9 2.4 2.0 2.5 2.5 3.3 2.3 N76D S163G Diff 1.1 3.7 0.1 2.95.1 2.7 1.6 N238E Q137H HSD 0.9 2.4 2.2 2.5 2.5 3.3 2.3 S141H R145HN238E L262E Diff 1.2 4.4 2.5 1.8 4.2 3.0 4.4 Q137H S141H HSD 0.9 2.4 2.02.5 2.5 3.5 2.3 R145H S3T N76D Diff 1.1 3.5 1.9 1.3 3.9 3.5 4.0 S156DY209W HSD 0.9 2.4 2.0 2.5 2.6 3.3 2.3 Q137H S141H R145H

Example 7: Wash Performance of Subtilase Variants

The wash performance of protease variants that have the same amino acidsequence as SEQ ID NO: 3 except for the substitutions as per the tablebelow on the stains as indicated; reference is the protease according toSEQ ID NO: 3. in laundry was assessed using the Automatic MechanicalStress Assay (AMSA), where the wash performance of many small volumeenzyme-detergent solutions can be examined. The AMSA plate has a numberof slots for test solutions and a lid that firmly squeezes the textileto be washed against the slot openings. During the wash, the plate, testsolutions, textile and lid were vigorously shaken to bring the testsolution in contact with the textile and apply mechanical stress in aregular, periodic, oscillating manner. For further description see WO02/42740 especially the paragraph “Special method embodiments” at pages23-24.

The laundry experiments were conducted under the experimental conditionsspecified in Table 5.

TABLE 5 Detergent dosage 2.0 g/L Test solution volume 160 μL (20 μLenzyme = 140 μL detergent) pH 8.4 Wash time 20 minutes Temperature 20°C. Water hardness 12° dH

Model detergent and test materials were as described in Table 1:

TABLE 6 Composition of model detergents and test materials DetergentLaundry liquid model detergent (Table 1) Test material C-05(Blood/milk/ink on cotton) PC-03 (Chocolate milk with carbon black onPolyester/cotton, 65/35) CS-37 (Full egg/pigment on cotton)

Test materials were obtained from Center For Testmaterials BV, 3133 KTVlaardingen, the Netherlands.

Water hardness was adjusted to 12° dH by addition of CaCl₂, MgCl₂, andNaHCO₃ (Ca²⁺:Mg²⁺=2:1:4.5) to the test system. After washing, thetextiles were flushed in tap water and dried.

The wash performance was measured as the brightness of the color of thetextile washed. Brightness can also be expressed as the intensity of thelight reflected from the sample when illuminated with white light. Whenthe sample is stained the intensity of the reflected light is lower thanthat of a clean sample. Therefore the intensity of the reflected lightcan be used to measure wash performance.

Color measurements were made with a Kodak iQsmart flatbed scanner(Kodak, Midtager 29, DK-2605 Brøndby, Denmark), which is used to capturean image of the washed textile.

To extract a value for the light intensity from the scanned images,24-bit pixel values from the image were converted into values for red,green and blue (RGB). The intensity value (Int) was calculated by addingthe RGB values together as vectors and then taking the length of theresulting vector:

Int=√{square root over (r ² +g ² +b ²)}

The results are shown in Table 7. The results are given as relativeperformance compared to SEQ ID NO: 3 at an enzyme concentration of 30 nMon three different swatches.

TABLE 7 AMSA relative performance of variants compared to SEQ ID NO: 3.Mutations compared Stain to SEQ ID NO 3 PC-03 C-05 CS-37 R45E 1.0 0.90.9 R45D 0.8 0.9 0.9 Q59D 1.0 1.0 1.1 S87E 1.1 1.0 1.2 N117E 1.1 0.9 0.8H120V 0.9 0.9 0.9 L124M 1.0 1.0 1.1 P129D 1.1 1.0 0.7 S156D 0.8 0.9 1.0S163G 1.1 1.2 1.0 S163A 0.9 1.0 1.0 N185Q 1.1 1.1 1.2 Y209W 0.8 0.9 1.0M222Q N.D. 0.9 3.0 V244T 1.0 1.0 1.0 N261D 0.9 0.9 1.4 N261T 1.0 1.1 1.1L262Q 1.0 1.0 1.1 L262E 1.0 1.0 1.4 Q59D + H120D 1.0 1.0 1.4 G61D + N76D1.0 0.9 1.6 S3T + N76D 1.1 1.0 1.5 S3T + H120D 1.1 0.9 0.9 G61D + H120D1.0 0.9 0.9 P55S + H120D 1.0 1.1 1.1 S163G + A228V 1.1 1.1 1.1 S163G +N261D 1.0 1.1 1.0 S3T + S163G 1.1 1.1 1.0 G61D + S163G 1.0 1.1 1.1S156D + S163G 0.9 1.1 1.3 Q59D + S163G 1.0 1.1 1.4 N76D + S163G 1.0 1.10.9 P55S + S163G 1.0 1.1 0.9 H120D + S163G 1.0 1.1 0.9 T58L + Q59D 1.21.0 1.6 P55S + T58L 1.0 1.0 0.9 T58L + G97D 0.9 1.0 1.3 T58L + S106A 1.11.0 0.9 T58L + A228V 1.1 1.0 0.9 S3T + T58L 1.0 1.0 1.1 T58L + S156D 1.21.1 1.5 T58L + Y91H 1.1 1.0 1.0 T58L + H120D 1.1 1.0 1.0 T58L + S163G1.2 1.0 0.7 S163G + N261D 1.0 0.9 1.3 T58L + N261D 0.9 1.0 0.9 T58L +N76D 1.0 1.0 1.0 S3T + N76D + H120D 1.0 0.9 1.3 S3T + N76D + A228V 1.30.9 1.2 S3T + N76D + S156D 0.9 0.9 2.2 S3T + N76D + Y209W 1.0 1.0 1.1S3T + N76D + Y209W + V244T 0.8 0.9 1.0

TABLE 8 Relative performance compared to SEQ ID NO: 3 at an enzymeconcentration of 30 nM on two different swatches. Stain PC-03 (Chocolatemilk with C-05 Mutations compared carbon black on (Blood/milk/ to SEQ IDNO 3 Polyester/cotton) ink on cotton) S3T 1.0 1.1 P55N 1.1 1.1 T58W 1.01.1 T58Y 1.1 1.0 T58W 1.1 1.1 Q59T 1.2 1.1 Q59N 1.1 1.0 Q59M 1.1 1.0Q59D 1.2 1.0 G61D 1.2 1.0 G97S 1.1 1.1 A98D 1.1 1.0 S106A 1.1 1.1 H120N1.1 1.1 H120K 1.0 1.1 H120D 1.3 1.1 S161T 1.0 0.9 S163G 1.4 1.1 N238H1.0 1.1 Y171L 1.0 1.0

The results show that the stabilized variants of SEQ ID NO 3 show on paror improved wash performance compared to the wash performance of SEQ IDNO 3.

Example 8: Result of Terg-O-Tometer (TOM) Wash Assay

The TOM wash experiment was conducted under the experimental conditionsspecified below:

TABLE 9 TOM wash conditions Detergent Liquid laundry model detergent(Table 1) Detergent dose 4.66 g/L pH pH was measured to be 7.6, but isused “as is” in the current detergent solution and is not adjusted.Water 16° dH, adjusted by adding CaCl₂*2H₂O, hardness MgCl₂*6H₂O andNaHCO₃ (5:1:3) to Milli-Q water. Enzyme conc. 60 nM Test solution 1000mL volume Test material 2 swatches, each 5 × 5 cm, of each of the 5soiled textile types per beaker (i.e. 10 soiled swatches per beaker):PC-03 (Chocolate-milk/ink on cotton/polyester) C-H010 (Cocoa, cooked upwith milk on cotton) C-05 (Blood/milk/soot on cotton) CS-01 (Aged bloodon cotton) CS-37 (Full egg/pigment on cotton) Cotton ballast swatches(50%:50% WFK10A (Standard Cotton Fabric, Unsoiled): WFK80A (cotton), 5 ×5 cm (wfk Testgewebe GmbH, Christenfeld 10; D-41379 Brijggen-Bracht;Germany) added to give a total weight of 30 g of soiled textileswatches + ballast per TOM beaker. Temperature 20° C. Wash time 30 minRinse time  5 min Rotation 120 rpm

TABLE 10 Relative performance compared to SEQ ID NO: 3 of subtilasevariants of having the indicated mutations compared to SEQ ID NO: 3.Delta remission PC-03 C-H010 C-05 CS-01 CS-37 R45E 1.4 1.0 1.2 1.9 1.1R45D 1.2 1.0 1.0 0.7 0.8 N117E 1.2 1.2 1.0 1.2 1.3 M222Q 0.7 0.9 1.1 0.91.4 L262E 1.3 1.1 1.1 1.2 1.9 P129D 1.3 0.8 1.1 0.8 1.3 S87E 1.2 1.1 1.22.2 1.0 Q59D + H120D 1.5 1.0 1.0 0.9 1.1 N76D + H120D 1.3 2.1 1.0 1.31.6 N76D + S156D 1.3 1.0 1.0 2.9 2.1 H120D + S156D 1.3 0.9 1.0 2.9 1.6G61D + N76D 1.1 1.0 1.1 1.1 2.3 R45E + L262E 1.4 2.2 1.0 1.5 1.2 Q59D +G61D 1.5 0.7 1.1 0.8 1.7 S87E + L262E 1.2 2.7 1.1 0.7 1.2 G61D + L262E1.4 2.3 1.0 0.9 1.3 Q59D + L262E 1.4 1.8 1.0 0.8 1.5 R45E + Q59D 1.3 1.10.9 1.0 1.4 Q59D + S156D 1.9 0.9 1.0 1.1 1.7 S156D + L262E 1.4 1.3 1.11.1 1.2 S163G + N238E + L262E 1.5 1.8 0.9 0.9 1.4 S3T + V4I + S163G +N261D 1.6 1.5 1.1 1.3 1.1 H120D + S163G + N261D 1.5 1.4 1.0 0.9 1.2Y91H + N117H + N238H 1.3 1.3 1.0 0.9 1.8 S3T + N76D + H120D 1.3 0.9 1.02.5 1.4 S3T + N76D + S156D 1.2 1.2 1.0 1.0 1.0 T58L + S163G + N261D 1.61.4 1.1 0.9 1.3 S3T + V4I + S163G + N261D 1.6 1.1 1.0 0.9 1.1 S87E +S163G + L262E 1.6 1.0 1.1 0.6 1.2 S156D + S163G + L262E 1.6 2.3 1.2 1.01.5 T58L + S163G + N261D 1.5 2.5 1.2 0.8 1.1 S156D + S163G + L262E 1.71.4 1.1 0.9 1.4 S3T + N76D + Y209W + 1.5 1.4 1.0 1.2 1.5 N261D + L262E

Example 9 Wash Performance of Protease Variants

Variants having the mutations as indicated in Table 15 compared to SEQID NO: 3 was washed under different conditions, AMSA wash with lowconcentration of protease (30 nM), AMSA wash with high concentration ofprotease (300 nM), TOM wash and full scale wash (FSW). The detergentdisclosed in Table 1 and the PC-03 (Chocolate-milk/ink oncotton/polyester) test materials were used for the washes. The resultswere compared with the performance of the reference protease having SEQID NO: 3 and the performance shown in the table 11 below where theresult for the reference protease is set to 1.00.

TABLE 11 AMSA AMSA (30 (300 Mutations compared to SEQ ID NO 3 nM) nM)TOM FSW N62D + H120D 1.44 1.39 H120D + N261D 1.52 1.38 N76D + N261D 1.34N76D + A228V + N261D 1.46 1.54 A194P + G195E + V205I + N261D 1.41 1.25R45E 1.06 1.43 1.43 N76D + H120D + N261D 1.13 1.18 H120D + S163G + N261D1.03 1.63 1.49 S3T + Q59D + N76D 1.15 S3T + N76D + H120D 1.1 1.31 S3T +N76D + A194P + G195E + 1.43 1.53 V199M + V205I S3T + N76D + S156D 1.021.23 S3T + N76D + Y209W + N261D 1.33 1.27 S3T + N76D + H120D + Y209W1.27 S3T + N76D + S156D + Y209W 1.31 1.38 S3T + V4I + N76D + A228V +N261D 1.34 S3T + V4I + N76D + H120D 1.32 H120D + P131F + A194P + N261D1.45 N76D + E136H + A228V + N261D 1.12 N76D + N2185 + A228V + N261D 1.01N76D + N218Q + A228V + N261D 1.01 1.45 1.46 N76D + N218A + A228V + N261D1.07 1.47 1.49 K27Q + R45E 1.32 N76D + A228V + L262E 1.77 1.38 R45E +A88S 1.06 1.58 1.26 S87E + K237E 1.24 N261D + L262E 1.41 S87E + L262E1.22 S87E + N238E 1.32 K27Q + S87E 1.53 N76D + N117E 1.34 H120D + N238E1.08 Q59D + L262E 1.26 K27Q + L262E 1.22 H120D + L262E 1.28 K27Q + Q59D1.13 K27Q + S156D 1.18 K27Q + G61D 1.21 Q59D + N261D 1.22 1.49 Q59D +N117E 1.19 K237E + N261D 1.11 Q59D + N238E 1.4 A15T + H120D + N261D 1.22N76D + S163G + N238E 1.18 H120D + S163G + L262E 1.15 H120D + S163G +N261D 1.08 1.18

1. A subtilase variant having at least 90% sequence identity to SEQ IDNO: 3 wherein the variant has a glutamic acid residue (E) in position101, wherein the subtilase variant further comprises one or moresubstitutions selected from the group consisting of S156D, L262E, Q137H,S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61D,R, S87E, G97S, A98D,E,R,S106A,W, N117E, H120V,D,K,N, S124M, P129D, E136Q, S143W, S161T, S163A,G,Y171L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D andL262N,Q,D, wherein the variant has increased stability in a liquiddetergent composition compared to the subtilase having the amino acidsequence of SEQ ID NO: 3 and wherein the positions corresponds to thepositions of SEQ ID NO:
 1. 2. The subtilase variant of claim 1, whereinthe subtilase further has improved wash performance compared with thesubtilase having SEQ ID NO:
 3. 3. The subtilase variant of claim 1,wherein the subtilase further has improved stability compared with thesubtilase having SEQ ID NO:
 3. 4. The subtilase variant of claim 1,wherein the subtilase variant comprises one or more substitutionsselected from the group consisting of: R45E,D,Q; T58L; G61D; S87E; G97S;A98E; S160A; N117E; H120D,K,V; P129D; E136Q; Q137H; S156D; S161T;S163A,G; V199M; M222Q; N261T and L262E,Q N.
 5. The subtilase variant ofclaim 1, wherein the variant is selected from the group consisting of:SEQ ID NO: 3+S3T, SEQ ID NO: 3+R45E,D SEQ ID NO: 3+P55N, SEQ ID NO:3+T58W,Y,L, SEQ ID NO: 3+Q59D,M,N,T, SEQ ID NO: 3+G61D,R, SEQ ID NO:3+S87E, SEQ ID NO: 3+G97S, SEQ ID NO: 3+A98D,E,R, SEQ ID NO: 3+S106A,W,SEQ ID NO: 3+N117E, SEQ ID NO: 3+H120V,D,K,N, SEQ ID NO: 3+S124M, SEQ IDNO: 3+P129D SEQ ID NO: 3+E136Q, SEQ ID NO: 3+S143W, SEQ ID NO: 3+S161T,SEQ ID NO: 3+S163A,G, SEQ ID NO. 3+Y171L, SEQ ID NO: 3+A172S, SEQ ID NO:3+N185Q, SEQ ID NO: 3+V199M, SEQ ID NO: 3+Y209W, SEQ ID NO: 3+M222Q, SEQID NO: 3+N238H, SEQ ID NO: 3+V244T, SEQ ID NO: 3+N261T, SEQ ID NO:3+L262N,Q,D,E SEQ ID NO: 3+N76D+S163G+N238E SEQ ID NO: 3+S156D+L262E SEQID NO: 3+N238E+L262E SEQ ID NO: 3+S3T+N76D+S156D+Y209W SEQ ID NO:3+H120D+S163G+N261D SEQ ID NO: 3+S163G+N128Q+N238E+L262E SEQ ID NO:3+K27Q+H120D+S163G+N261D SEQ ID NO: 3+V104T+H120D+S156D+L262E SEQ ID NO:3+G195E+V199M SEQ ID NO: 3+S3T+V4I+N261D SEQ ID NO:3+A194P+G195E+V199M+V205I SEQ ID NO: 3+H120D+A228V SEQ ID NO:3+S3T+V4I+A228V SEQ ID NO: 3+H120D+N261D SEQ ID NO: 3+H120D+S163G+N261DSEQ ID NO: 3+N76D+A228V+L262E SEQ ID NO:3+N76D+Q137H+S141H+R145H+S163G+N238E SEQ ID NO:3+Q137H+S141H+R145H+N238E+L262E SEQ ID NO:3+S3T+N76D+Q137H+S141H+R145H+S156D+Y209W SEQ ID NO:3+H120D+Q137H+S141H+R145H+S163G+N261D SEQ ID NO:3+N76D+Q137H+5141H+R145H+A228V+N261D SEQ ID NO:3+A194P+G195E+V199M+V205I+A228V+N261D SEQ ID NO: 3+N62D+H120D SEQ ID NO:3+H120D+N261D SEQ ID NO: 3+N76D+N261D SEQ ID NO: 3+N76D+A228V+N261D SEQID NO: 3+A194P+G195E+V205I+N261D SEQ ID NO: 3+N76D+H120D+N261D SEQ IDNO: 3+H120D+S163G+N261D SEQ ID NO: 3+S3T+Q59D+N76D SEQ ID NO:3+S3T+N76D+H120D SEQ ID NO: 3+S3T+N76D+A194P+G195E+V199M+V205I SEQ IDNO: 3+S3T+N76D+S156D SEQ ID NO: 3+S3T+N76D+Y209W+N261D SEQ ID NO:3+S3T+N76D+H120D+Y209W SEQ ID NO: 3+S3T+N76D+S156D+Y209W SEQ ID NO:3+S3T+V4I+N76D+A228V+N261D SEQ ID NO: 3+S3T+V4I+N76D+H120D SEQ ID NO:3+H120D+P131F+A194P+N261D SEQ ID NO: 3+N76D+E136H+A228V+N261D SEQ ID NO:3+N76D+N218S+A228V+N261D SEQ ID NO: 3+N76D+N218Q+A228V+N261D SEQ ID NO:3+N76D+N218A+A228V+N261D SEQ ID NO: 3+K27Q+R45E SEQ ID NO:3+N76D+A228V+L262E SEQ ID NO: 3+R45E+A88S SEQ ID NO: 3+S87E+K237E SEQ IDNO: 3+N261D+L262E SEQ ID NO: 3+S87E+L262E SEQ ID NO: 3+S87E+N238E SEQ IDNO: 3+K27Q+S87E SEQ ID NO: 3+N76D+N117E SEQ ID NO: 3+H120D+N238E SEQ IDNO: 3+Q59D+L262E SEQ ID NO: 3+K27Q+L262E SEQ ID NO: 3+H120D+L262E SEQ IDNO: 3+K27Q+Q59D SEQ ID NO: 3+K27Q+S156D SEQ ID NO: 3+K27Q+G61D SEQ IDNO: 3+Q59D+N261D SEQ ID NO: 3+Q59D+N117E SEQ ID NO: 3+K237E+N261D SEQ IDNO: 3+Q59D+N238E SEQ ID NO: 3+A15T+H120D+N261D SEQ ID NO:3+N76D+S163G+N238E SEQ ID NO: 3+H120D+S163G+L262E SEQ ID NO:3+H120D+S163G+N261D SEQ ID NO: 3+Q59D+H120D SEQ ID NO: 3+G61D+N76D SEQID NO: 3+S3T+N76D SEQ ID NO: 3+S3T+H120D SEQ ID NO: 3+G61D+H120D SEQ IDNO: 3+P55S+H120D SEQ ID NO: 3+S163G+A228V SEQ ID NO: 3+S163G+N261D SEQID NO: 3+S3T+S163G SEQ ID NO: 3+G61D+S163G SEQ ID NO: 3+S156D+S163G SEQID NO: 3+Q59D+S163G SEQ ID NO: 3+N76D+S163G SEQ ID NO: 3+P55S+S163G SEQID NO: 3+H120D+S163G SEQ ID NO: 3+T58L+Q59D SEQ ID NO: 3+P55S+T58L SEQID NO: 3+T58L+G97D SEQ ID NO: 3+T58L+S106A SEQ ID NO: 3+T58L+A228V SEQID NO: 3+S3T+T58L SEQ ID NO: 3+T58L+S156D SEQ ID NO: 3+T58L+Y91H SEQ IDNO: 3+T58L+H120D SEQ ID NO: 3+T58L+S163G SEQ ID NO: 3+S163G+N261D SEQ IDNO: 3+T58L+N261D SEQ ID NO: 3+T58L+N76D SEQ ID NO: 3+S3T+N76D+H120D SEQID NO: 3+S3T+N76D+A228V SEQ ID NO: 3+S3T+N76D+S156D SEQ ID NO:3+S3T+N76D+Y209W SEQ ID NO: 3+S3T+N76D+Y209W+V244T SEQ ID NO:3+N76D+H120D SEQ ID NO: 3+N76D+S156D SEQ ID NO: 3+H120D+S156D SEQ ID NO3+R45E+L262E SEQ ID NO 3+Q59D+G61D SEQ ID NO 3+S87E+L262E SEQ ID NO3+G61D+L262E SEQ ID NO 3+Q59D+L262E SEQ ID NO 3+R45E+Q59D SEQ ID NO3+Q59D+S156D SEQ ID NO 3+S156D+L262E SEQ ID NO 3+S163G+N238E+L262E SEQID NO 3+S3T+V4I+S163G+N261D SEQ ID NO 3+H120D+S163G+N261D SEQ ID NO3+Y91H+N117H+N238H SEQ ID NO 3+T58L+S163G+N261D SEQ ID NO3+S3T+V4I+S163G+N261D SEQ ID NO 3+S87E+S163G+L262E SEQ ID NO3+S156D+S163G+L262E SEQ ID NO 3+T58LS163G+N261D SEQ ID NO3+S156DS163G+L262E and SEQ ID NO 3+S3T+N76D+Y209W+N261D+L262E.
 6. Anucleotide sequence encoding a variant according to claim
 1. 7. Anexpression vector comprising the nucleotide sequence of claim
 6. 8. Arecombinant host cell comprising the nucleotide sequence of claim 6 orthe expression vector of claim
 7. 9. A method for producing the variantaccording to claim 1, comprising: a. Providing a recombinant host cellof claim 8; b. Culturing the recombinant host cell under conditionsleading to expression of the variant; and c. Isolating the variant. 10.The method of claim 1, wherein the variant has at least 95% sequenceidentity to SEQ ID NO:
 3. 11. The method of claim 1, wherein the varianthas at least 96% sequence identity to SEQ ID NO:
 3. 12. The method ofclaim 1, wherein the variant has at least 97% sequence identity to SEQID NO:
 3. 13. The method of claim 1, wherein the variant has at least98% sequence identity to SEQ ID NO: 3.